Oligosaccharide enzyme substrates and inhibitors: methods and compositions

ABSTRACT

Oligosacaharide compounds that are substrates and inhibitors of glycosyltransferase and glycosidase enzymes and compositions containing such compounds are disclosed. A method of glycosylation is also disclosed. An E. coli transformed with phagemid CMPSIL-1, which phagemid comprises a gene for a modified CMP-sialic acid synthetase enzyme, which transformed E. coli has the ATCC accession No. 68531 is also provided.

CROSS-REFERENCE TO COPENDING APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.08/219,242, filed Mar 29, 1994, now U.S. Pat. No. 5,461,143, which is acontinuation-in-part of U.S. patent application Ser. No. 07/852,409,filed Mar. 16, 1992, now abandoned, which is itself acontinuation-in-part of U.S. patent application Ser. No. 07/738,211filed Jul. 30, 1991, now abandoned, which is itself acontinuation-in-part of U.S. patent application Ser. No. 07/670,701filed Mar. 18, 1991 now U.S. Pat. No. 5,278,299, and U.S. patentapplication Ser. No. 07/707,600 filed May 30, 1991, now abandoned.

TECHNICAL FIELD

The present invention relates to oligosaccharide compounds, and moreparticularly to di-, tri- and tetrasaccharides that are substrates orinhibitors of glycosyltransferase and glycosidase enzymes, theirmanufacture and use.

BACKGROUND ART

The stereocontrolled synthesis of oligosaccharides based onsophisticated protection/deprotection, activation and couplingstrategies has been well established. See, e.g., Danishefsky et al. J.Am. Chem. Soc., 111:6656 (1989); Okamoto et al., Tetrahedron, 46:5835(1990); and Ito et al., Tetrahedron 46:89 (1990). A useful alternativeto as the major planes have a problem in that their intrinsicdesensitization due to dyes is great enzymes. Toone et al., Tetrahedron,45:5365 (1989). One advantage of such enzymatic synthesis is the lack ofextensive protection and deprotection steps. A disadvantage of suchenzymatic synthesis is the apparent limitation of product formation thatresults from the specificity of glycosyltransferase and glycosidaseenzymes.

Glycosyltransferases are highly specific enzymes that catalyze thetransfer or activated donor monosaccharides to acceptor saccharides.That transfer results in the elongation or synthesis of an oligo- orpolysaccharide.

A number of glycosyltransferase types have been described includingsialyltransferases, N-acetylgalactosaminyltransferases,N-acetylglucos-aminyltransferases and the like. Beyer, et al., Adv.Enzymol., 52:23 (1981). The designation of those enzymes indicates thenature of the donor substrate. Thus, for example, a sialyltransferasetransfers a sialic acid moiety to an acceptor molecule.

Within each of the general enzyme types set forth above, specifictransferase enzymes are additionally designated by the type ofglycosidic linkage formed. For example, a β1,4-galactosyltransferasetransfers a galactosyl moiety to an acceptor molecule, forming aβ1,4-glycosidic linkage with such acceptor.

Further, glycosyltransferases are characterized by the acceptor to whichthe donor glycosyl compound is transferred. A β1,4-galactosyltransferasefrom bovine milk (GalT, EC 2.4.1.22) is known to acceptN-acetylglucosamine (GlcNAc) and its glycosides (βis better thanα-glycoside) as acceptor substrates. See, e.g., Schanbacher, et al., J.Biol. Chem., 245:5057 (1970); Berliner, et al., Mol. Cell. Biochem.,62:37 (1984); Nunez, et al., Biochemistry, 19:495 (1980); Beyer, et al.,Adv. Enzymol., 52:23 (1981); Barker, et al., J. Biol. Chem., 247:7135(1972); and Babad, et al., J. Biol. Chem., 241:2672 (1966). Glucose andits α-and β-glucosides are also acceptable; however, lactalbumin isrequired for α-glucosides. Beyer, et al., supra.

Taken together with the donor and linkage specificity set forth above,such acceptor specificity is used to define unique products ofglycosyltransferase activity.

Oligosaccharides are considered to have a reducing end and anon-reducing end, whether or not the saccharide at the reducing end isin fact a reducing sugar. In accordance with accepted nomenclature,oligosaccharides are depicted herein with the non-reducing end on theleft and the reducing end on the right.

All oligosaccharides described herein are, thus, described with the nameor abbreviation for the non-reducing saccharide (i.e., Gal), followed bythe configuration of the glycosidic bond (α or β), the ring position ofthe non-reducing saccharide involved in the bond (1 or 2), the ringposition of the reducing saccharide involved in the bond (2, 3, 4, 6 or8), and then the name or abbreviation of the reducing saccharide (i.e.,GlcNAc).

It is often extremely difficult to make synthetic saccharides that canbe used to study naturally occurring synthetic routes by inhibiting thesynthetic reactions. The lack of such synthetic inhibitors hampersattempts to investigate the effects of metabolic changes on carbohydrateproduction and turnover.

It is also often difficult to prepare novel, non-naturally occurringoligo- and polysaccharides that are useful as carriers or solubilizingagents for drugs and, which because of their non-natural structures, areresistant to degradation in vivo.

There is, therefore, a pressing need for oligosaccharide compounds andefficient methods of making the same that serve as substrates orinhibitors of transferase and glycosidase enzymes.

BRIEF SUMMARY OF THE INVENTION

The present invention provides novel oligosaccharides that aresubstrates for some glycosyltransferases and that inhibit otherglycosyltransferase and glycosidase enzymes as well as a method formaking such oligosaccharides. Those oligosaccharides are also useful asbuilding blocks in the synthesis of other oligosaccharides such assialyl Le^(x) and its analogs.

In one aspect, the present invention contemplates an oligosaccharidethat corresponds to structural Formula I: ##STR1## wherein X is O, S,SO, SO₂ or NR₁₆, wherein R₁₆ is hydrogen, C₁ -C₁₂ acyl, C₁ -C₁₂ alkyl,C₁ -C₄ alkoxycarbonyl or >NR₁₆ is a C₁ -C₁₂ alkyl N-oxide;

R₁ is absent, hydrogen, hydroxyl, C₁ -C₄ acyl, C₁ -C₄ alkoxycarbonyloxy,a saturated or unsaturated alkoxide or alkoxy alkoxide containing up tofive carbon atoms or a glycosidially linked saccharide;

R₁ ' is hydrogen or R₁ R₁ ' together form an oxo group;

R₂ is absent, hydrogen, hydroxyl, halide, C₁ -C₅ alkoxy or NR₁₇ R₁₈where R₁₇ is hydrogen or C₁ -C₄ alkyl and R₁₈ is hydrogen, C₁ -C₄ alkyl,C₁ -C₄ acyl, or C₁ -C₄ alkoxycarbonyl, or NR₁₇ R₁₈ together from acyclic imido group containing 4-8 carbon atoms;

R₃ and R₄ are independently hydrogen, C₁ -C₄ alkyl, hydroxyl,thiophenyl, C₁ -C₃ alkylthio, a saturated or unsaturated alkoxide oralkoxy alkoxide containing up to five carbon atoms, a glycosidicallylinked glucosyl, N-acetylglucosaminyl, galactosyl,N-acetylgalactosaminyl, fucosyl, mannosyl, rhamnosyl, sialyl group or adisaccharide thereof, or R₃ and R₄ together form an oxo group, with theproviso that at least one of R₃ and R₄ is hydrogen except when (i) R₃and R₄ together form an oxo group, (ii) R₂ and R₃ are absent with theirbonds forming ethylenic unsaturation or (iii) X is NR₁₆ ;

R₅ is absent, hydrogen, hydroxyl, methyl, C₁ -C₄ acyl or C₁ -C₄alkoxycarbonyloxy;

R₆ is absent, hydroxymethyl, methyl, trihydroxyropyl, methylene C₁ -C₄acyloxy or benzyloxy;

R₇ is hydrogen or carboxyl;

R₈ is hydrogen, hydroxyl or acetamido;

R₉ is hydroxymethyl, methyl, trihydroxypropyl, methylene C₁ --C₄ acyloxyor benzyloxy, and 3-acetoxy-1,2-dihydroxypropyl,3-lactyloxy-1,2-dihydroxypropyl, 3-azido-1,2-dihydroxypropyl, and3-fluoro-1,2-dihydroxypropyl when R₈ is hydrogen and R₁₁ isN-acetylamino;

R₁₀ is absent, hydroxyl or acetamido;

R₁₁ is absent, hydroxyl or acetamido;

R₁₂ is hydroxyl or acetamido;

R₁₃ is hydroxymethyl or trihydroxypropyl, and3-acetoxy-1,2-dihydroxypropyl, 3-lactyloxy-1,2-dihydroxypropyl,3-azido-1,2-dihydroxypropyl, and 3-fluoro-1,2-dihydroxypropyl when R₁₅is hydrogen and R₁₂ is N-acetylamino;

R₁₄ is hydrogen or carboxyl;

R₁₅ is hydrogen, hydroxyl or acetamido; and

m is zero or one such that when m is zero, ring C is absent and when mis one, ring C is present;

with the provisos (a) that one of substituents R₁, R₂ and R₅ or ahydroxyl group of R₆ is absent from ring B and ring B is joined to ringA through a glycosidic bond to the ring B carbon of the absentsubstituent, and that a numbered substituent or hydroxyl is only absentwhen ring A is joined to ring B at the position of that substituent orhydroxyl except as enumerated herein; (b) that when m is one, one ofsubstituents R₁₀ and R₁₁ or a hydroxyl group of R₉ is absent from ring Aand ring C is joined to ring A through a glycosidic bond to the ring Acarbon of the absent substituent or hydroxyl, and that numberedsubstituent or hydroxyl is only absent when ring c is joined to ring Aat the position of that substituent or hydroxyl, or a second of R₁, R₂,R₅ or a hydroxyl of R₆ is absent and ring C is joined to ring B througha glycosidic bond to the ring B carbon of the second absent substituentor hydroxyl; (c) that X is O only if one of the following structures ispresent; (i) R₁ and R₁ ' together form an oxo group, (ii) R₁ and eitherR₃ or R₄ are not hydroxyl, (iii) R₃ and R₄ together form an oxo group,(iv) either R₃ and R₄ is C₁ -C₃ alkylthio, or (v) R₂ and R₃ are absentand their bonds form ethylenic unsaturation and either R₁, R₅, R₈ or R₉is not hydroxyl or R₆ is not hydroxymethyl; and (d) that R₂ and R₃ areabsent and their bonds form ethylenic unsaturation only when X is O.

In another aspect, the present invention contemplates an oligosaccharidethat corresponds to structural Formula II: ##STR2## wherein X is O, S,SO, SO₂ or NR₁₆, wherein R₁₆ is hydrogen, C₁ -C₁₂ acyl, C₁ -C₁₂ alkyl,C₁ -C₄ alkoxycarbonyl or >NR₁₆ is a C₁ -C₁₂ alkyl N-oxide;

R₁ is absent, hydrogen, hydroxyl, C₁ -C₄ acyl, C₁ -C₄ alkoxycarbonyloxy,a saturated or unsaturated alkoxide or alkoxy alkoxide containing up to5 carbon atoms or a glycosidically linked saccharide;

R₁ ' is, hydrogen or R₁ and R₁ ' together form an oxo group;

R₂ is absent, hydrogen, hydroxyl, halide, C₁ -C₅ alkoxy or NR₁₂ R₁₈where R₁₇ is hydrogen or C₁ -C₄ alkyl and R₁₈ is hydrogen, C₁ -C₄ alkyl,C₁ -C₄ acyl, or C₁ -C₄ alkoxycarbonyl, or NR₁₇ R₁₈ together form acyclic imido group containing 4-8 carbon atoms;

R₃ and R₄ are independently hydrogen, C₁ -C₄ alkyl, hydroxyl,thiophenyl, C₁ -C₃ alkylthio, a saturated or unsaturated alkoxide oralkoxy alkoxide containing up to 5 carbon atoms, a glycosidically linkedglucosyl, N-acetylglucosaminyl, galactosyl, N-acetylgalactosaminyl,fucosyl, mannosyl, rhamnosyl, sialyl group or a disaccharide thereof, orR₃ and R₄ together form an oxo group, with the proviso that at least oneof R₃ and R₄ is hydrogen except when (i) R₃ and R₄ together from an oxogroup, (ii) R₂ and R₃ are absent with their bonds forming ethylenicunsaturation or (iii) X is NR₁₆ ;

R₅ is absent, hydrogen, hydroxyl, methyl, C₁ -C₄ acyl or C₁ -C₄alkoxycarbonyloxy;

R₆ is absent, hydroxymethyl, methyl, trihydroxypropyl, methylene C₁ -C₄acyloxy or benzyloxy;

R₇ is hydrogen or carboxyl;

R₈ is absent, hydroxyl or acetamido;

R₉ is hydroxymethyl, methyl, trihydroxypropyl, methylene C₁ -C₄ acyloxyor benzyloxy, and 3-acetoxy-1,2-dihydroxypropyl,3-lactyloxy-1,2-dihydroxypropyl, 3-azido-1,2-dihydroxypropyl, and3-fluoro-1,2-dihydroxypropyl when R₈ is hydrogen and R₁₁ isN-acetylamino;

R¹¹ is hydroxyl or acetamido; with the provisos (a) that one ofsubstituents R₁, R₂, R₅ or a hydroxyl group of R₆ is absent from ring Band ring B is joined to ring A through a glycosidic bond to the ring Bcarbon of the absent substituent, and that a numbered substituent orhydroxyl is only absent when ring A is joined to ring B at the positionof that substituent or hydroxyl except as enumerated herein; (b) that Xis O only if one of the following structures is present; (i) R₁ and R₁ 'together form an oxo group, (ii) R₁ and either R₃ or R₄ are nothydroxyl, (iii) R₃ and R₄ together form an oxo group, (iv) either R₃ orR₄ is C₁ -C₃ alkylthio, or (v) R₂ and R₃ are absent and their bonds formethylenic unsaturation and either R₁, R₅, R₈ or R₉ is not hydroxyl or R₆is not hydroxymethyl; and (c) that R₂ and R₃ are absent and their bondsform ethylenic unsaturation only when X is O.

In another aspect, the present invention provides a method ofglycosylation that comprises the steps of admixing in an aqueous mediuman activated donor monosaccharide with an acceptor saccharide, ofFormula II, above, or of Formula III below, in the presence of acatalytic amount of a glycosyltransferase having specificity for boththe activated donor monosaccharide and the acceptor saccharide to form areaction mixture: ##STR3## wherein X and R₁₋₆ of Formula III are asdefined in Formula II above, and maintaining the reaction mixture for atime period and under conditions sufficient for the acceptor saccharideto be glycosylated and-form a glycosylated acceptor saccharide.

In a preferred embodiment, the glycosylation method comprises the stepsof:

(a) admixing in the presence of each other in an aqueous medium

(i) an acceptor saccharide;

(ii) a donor monosaccharide;

(iii) an activating nucleotide having specificity for the donormonosaccharide;

(iv) an activated donor monosaccharide regenerating system;

(v) a pyrophosphate scavenger; and

(vi) catalytic amounts of a glycosyltransferase having specificity forboth the activated form of the donor monosaccharide and the acceptorsaccharide and a nucleotide-sugar-pyrophosphorylase having specificityfor both the donor monosaccharide and the activating nucleotide to forma reaction mixture; and

(b) maintaining the reaction mixture for a time period and underconditions sufficient for the acceptor saccharide to be glycosylated andform a glycosylated acceptor saccharide.

The acceptor saccharide can be an acceptor monosaccharide or an acceptoroligosaccharide. The acceptor oligosaccharide can itself be prepared inthe reaction mixture, which reaction mixture further includes:

(a) a second acceptor saccharide;

(b) a second donor monosaccharide;

(c) a second activating nucleotide that has specificity for the seconddonor monosaccharide;

(d) a second activated donor monosaccharide regenerating system; and

(e) catalytic amounts of (i) a glycosyltransferase having specificityfor both the activated form of the second donor monosaccharide and thesecond acceptor saccharide and (ii) a nucleotide-sugar-pyrophosphorylasehaving specificity for both the second donor monosaccharide and thesecond activating nucleotide.

The activated donor monosaccharide regenerating system used in theglycosylation method comprises a phosphate donor and a catalytic amountof a kinase that catalyzes the transfer of phosphate from the phosphatedonor to an activating nucleotide.

The present invention also contemplates an E. coli transfected withphagemid CMPSIL-1, which phagemid comprises a gene encoding a modifiedCMP-sialic acid synthetase enzyme. That transformed E. coli has the ATCCaccession No. 68531. Phagemid CMPSIL-W10 and E. coli transfected withthat phagemid are also contemplated.

The present invention still further contemplates an acceptor saccharideof Formula III above.

Also contemplated by the present invention is a composition thatcomprises a glycosyltransferase or glycosidase inhibiting amount of abefore-described oligosaccharide compound dispersed in an aqueousmedium. The aqueous medium is preferably pharmaceutically acceptable.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings that form a portion of the specification:

FIG. 1 is a schematic diagram of plasmid pIN-GT showing the location ofthe GalT gene as well as other components including the DNA of SEQ IDNO:7 and corresponding amino acid residues of SEQ ID NO:8.

FIG. 2 is a schematic diagram showing the construction of the DNA insertcontaining the 1.3 kb PCR amplification product that includes theCMP-NeuAc synthetase structural gene as well as the upstream Lac Zpromoter and linking DNA having an Eco RI restriction site (underlined),a ribosome binding site (Rib, sequence underlined) and an ATG(underlined) start signal (SEQ ID NO:9), and the downstream amino acidresidue sequence of the tag peptide and DNA stop signal; SEQ ID NO:10(boxed), followed downstream by Xba I and Not I restriction sites, andthe arms from lambda Lcl vector according to Example 2.

FIG. 3 is a schematic diagram showing the major features of phagemidCMPSIL-1. The PCR amplification product insert from FIG. 2 is shown atthe top. The orientations of the insert and other genes are also shown.

DETAILED DESCRIPTION OF THE INVENTION

The Compounds

A compound of the invention is an oligosaccharide, i.e., a compoundcontaining two to ten saccharide units, and, preferably a disaccharide,trisaccharide or tetrasaccharide.

In one embodiment, an oligosaccharide of the present inventioncorresponds to structural Formula I: ##STR4## wherein X is O, S, SO, SO₂or NR₁₆, wherein R₁₆ is hydrogen, C₁ -C₁₂ acyl, C₁ -C₁₂ alkyl, C₁ -C₄alkoxycarbonyl or >NR₁₆ is a C₁ -C₁₂ alkyl N-oxide;

R₁ is absent, hydrogen, hydroxyl, C₁ -C₄ acyl, C₁ -C₄ alkoxycarbonyloxy,a saturated or unsaturated alkoxide or alkoxy alkoxide containing up tofive carbon atoms or a glycosidially linked saccharide;

R₁ ' is hydrogen or R₁ and R₁ ' together form an oxo group;

R₂ is absent, hydrogen, hydroxyl, halide, C₁ -C₅ alkoxy or NR₁₇ R₁₈where R₁₇ is hydrogen or C₁ -C₄ alkyl and R₁₈ is hydrogen, C₁ -C₄ alkyl,C₁ -C₄ acyl, or C₁ -C₄ alkoxycarbonyl, or NR₁₇ R₁₈ together form acyclic imido group containing 4-8 carbon atoms;

R₃ and R₄ are independently hydrogen, C₁ -C₄ alkyl, hydroxyl,thiophenyl, C₁ -C₃ alkylthio, a saturated or unsaturated alkoxide oralkoxy alkoxide containing up to five carbon atoms, a glycosidicallylinked glucosyl, N-acetylglucosaminyl, galactosyl,N-acetylgalactosaminyl, fucosyl, mannosyl, rhamnosyl, sialyl group or adisaccharide thereof, or R₃ and R₄ together form an oxo group, with theproviso that at least one of R₃ and R₄ is hydrogen except when (i) R₃and R₄ together form an oxo group, (ii) R₂ and R₃ are absent with theirbonds forming ethylenic unsaturation or (iii) X is NR₁₆ ;

R₅ is absent, hydrogen, hydroxyl, methyl, C₁ -C₄ acyl or C₁ -C₄alkoxycarbonyloxy;

R₆ is absent, hydroxymethyl, methyl, trihydroxyropyl, methylene C₁ -C₄acyloxy or benzyloxy;

R₇ is hydrogen or carboxyl;

R₈ is hydrogen, hydroxyl or acetamido;

R₉ is hydroxymethyl, methyl, trihydroxypropyl, methylene C₁ -C₄ acyloxyor benzyloxy, and 3-acetoxy-1,2-dihydroxypropyl,3-lactyloxy-1,2-dihydroxypropyl, 3-azido-1,2-dihydroxypropyl, and3-fluoro-1,2-dihydroxypropyl when R₈ is hydrogen and R₁₁ isN-acetylamino;

R₁₀ is absent, hydroxyl or acetamido;

R₁₁ is absent, hydroxyl or acetamido;

R₁₂ is hydroxyl or acetamido;

R₁₃ is hydroxymethyl or trihydroxypropyl, and3-acetoxy-1,2-dihydroxypropyl, 3-lactoyloxy-1,2-dihydroxypropyl,3-azido-1,2-dihydroxypropyl, and 3-fluoro-1,2-dihydroxypropyl when R₁₅is hydrogen and R₁₂ is N-acetylamino;

R₁₄ is hydrogen or carboxyl;

R₁₅ is hydrogen, hydroxyl or acetamido; and

m is zero or one such that when m is zero, ring C is absent and when mis one, ring C is present; and

with the provisos (a) that one of substituents R₁, R₂ and R₅ or ahydroxyl group of R₆ is absent from ring B and ring B is joined to ringA through a glycosidic bond to the ring B carbon of the absentsubstituent, and that a numbered substituent or hydroxyl is only absentwhen ring A is joined to ring B at the position of that substituent orhydroxyl except as enumerated herein; (b) that when m is one, one ofsubstituents R₁₀ and R₁₁ or a hydroxyl group of R₉ is absent from ring Aand ring C is joined to ring A through a glycosidic bond to the ring Acarbon of the absent substituent or hydroxyl, and that numberedsubstituent or hydroxyl is only absent when ring C is joined to ring Aat the position of that substituent or hydroxyl, or a second of R₁, R₂,R₅ or a hydroxyl of R₆ is absent and ring C is joined to ring B througha glycosidic bond to the ring B carbon of the second absent substituentor hydroxyl; (c) that X is O only if one of the following structures ispresent; (i) R₁ and R₁ ' together form an oxo group, (ii) R₁ and eitherR₃ or R₄ are not hydroxyl, (iii) R₃ and R₄ together form an oxo group,(iv) either R₃ and R₄ is C₁ -C₃ alkylthio, or (v) R₂ and R₃ are absentand their bonds form ethylenic unsaturation and either R₁, R₅, R₈ or R₉is not hydroxyl or R₆ is not hydroxymethyl; and (d) that R₂ and R₃ areabsent and their bonds form ethylenic unsaturation only when X is O.

Another oligosaccharide compound of the present invention thatcorresponds to structural Formula II ##STR5## wherein X is O, S, SO, SO₂or NR₁₆, wherein R₁₆ is hydrogen, C₁ -C₁₂ acyl, C₁ -C₄ alkyl, C₁ -C₄alkoxycarbonyl or >NR₁₆ is a C₁ -C₁₂ alkyl N-oxide;

R₁ is absent, hydrogen, hydroxyl, C₁ -C₄ acyl, C₁ -C₄ alkoxycarbonyloxy,a saturated or unsaturated alkoxide or alkoxy alkoxide containing up to5 carbon atoms or a glycosidically linked saccharide;

R₁ ' is hydrogen or R₁ and R₁ ' together form an oxo group;

R₂ is absent, hydrogen, hydroxyl, halide, C₁ -C₅ alkoxy or NR₁₂ R₁₈where R₁₇ is hydrogen or C₁ -C₄ alkyl and R₁₈ is hydrogen, C₁ -C₄ alkyl,C₁ -C₄ acyl, or C₁ -C₄ alkoxycarbonyl, or NR₁₇ R₁₈ together form acyclic imido group containing 4-8 carbon atoms;

R₃ and R₄ are independently hydrogen, C₁ -C₄ alkyl, hydroxyl,thiophenyl, C₁ -C₃ alkylthio, a saturated or unsaturated alkoxide oralkoxy alkoxide containing up to 5 carbon atoms, a glycosidically linkedglucosyl, N-acetylglucosaminyl, galactosyl, N-acetylgalactosaminyl,fucosyl, mannosyl, rhamnosyl, sialyl group or a disaccharide thereof, orR₃ and R₄ together form an oxo group, with the proviso that at least oneof R₃ and R₄ is hydrogen except when (i) R₃ and R₄ together from an oxogroup, (ii) R₂ and R₃ are absent with their bonds forming ethylenicunsaturation or (iii) X is NR₁₆ ;

R₅ is absent, hydrogen, hydroxyl, methyl, C₁ -C₄ acyl or C₁ -C₄alkoxycarbonyloxy;

R₆ is absent, hydroxymethyl, methyl, trihydroxypropyl, methylene C₁ -C₄acyloxy or benzyloxy;

R₇ is hydrogen or carboxyl;

R₈ is absent, hydroxyl or acetamido;

R₉ is hydroxymethyl, methyl, trihydroxypropyl, methylene C₁ -C₄ acyloxyor benzyloxy, and 3-acetoxy-1,2-dihydroxypropyl,3-lactyloxy-1,2-dihydroxypropyl, 3-azido-1,2-dihydroxypropyl, and3-fluoro-1,2-dihydroxypropyl when R₈ is hydrogen and R₁₁ isN-acetylamino;

R¹¹ is hydroxyl or acetamido;

with the provisos (a) that one of substituents R₁, R₂, R₅ or a hydroxylgroup of R₆ is absent from ring B and ring B is joined to ring A througha glycosidic bond to the ring B carbon of the absent substituent, andthat a numbered substituent or hydroxyl is only absent when ring A isjoined to ring B at the position of that substituent or hydroxyl exceptas enumerated herein; (b) that X is O only if one of the followingstructures is present; (i) R₁ and R₁ ' together form an oxo group, (ii)R₁ and either R₃ or R₄ are not hydroxyl, (iii) R₃ and R₄ together forman oxo group, (iv) either R₃ or R₄ is C₁ -C₃ alkylthio, or (v) R₂ and R₃are absent and their bonds form ethylenic unsaturation and either R₁,R₅, R₈ or R₉ is not hydroxyl or R₆ is not hydroxymethyl; and (c) that R₂and R₃ are absent and their bonds form ethylenic unsaturation only whenX is O.

Exemplary saccharide (sugar) units contain six-membered rings andinclude substituent configurations of common, naturally occurring sugarssuch as glucose (Glc), N-acetylglucosamine (GlcNAc), galactose (Gal),N-acetylgalactosamine (GalNAc), mannose (Man), rhamnose (Rha), fucose(Fuc), sialic acid (NeuAc), and the like, and their 2-deoxy derivatives.

The saccharide units are joined together by a glycosidic bond.Typically, the glycosidic bond is between the carbon atom at position 1of the non-reducing end sugar (ring A of Formulas I or II) and thecarbon atom at position 2, 3, 4 or 6 of the reducing end sugar (ring Bof Formulas I or II). When the non-reducing end sugar is sialic acid,the glycosidic bond is between the carbon atom at position 2 of sialicacid and the carbon atom at positions 2, 3, 4, 6 or 8 of the reducingend sugar.

The glycosidic bonding can have an α or a β configuration. β1,4-, α1,3-and α2,6-bonding are used as exemplary herein, and are preferred. Otherbond configurations are also contemplated.

In the above formulas, and in the other formulas utilized herein,typically only one group at each of the ring carbon atoms is shown. Thefourth, unshown group bonded to each of those ring carbons is a hydrogenatom, as would be present in an unsubstituted carbohydrate. The twoshown "fourth groups" are so shown to permit an oxo group to be present.Additionally, when R₃ and R₄ are hydrogen and hydroxyl, both anomers arecontemplated.

The above structural formulas and of those set forth hereinafter also donot show the orientation of groups R₁₋₁₅ relative to the plane of thering. Each of the α- and β-orientations is contemplated for each ofR₁₋₁₅, so those substituent groups are shown generally.

The orientation of a substituent is a function of the precursormolecule, and the substituent orientation can be varied as desired. Aswill be discussed hereinafter, particular orientations of R₁₋₁₅ arepreferred.

In regard to Formula I, it is first noted that ring C is present when mis one and absent when m is zero. Thus, when m is zero, Formula Ireduces to Formula II. When m is one, ring C can be bonded to ring A(Schemes 1 and 2) or to ring B (Scheme 3 and Table 1a) so that a linearor branched oligosaccharide, respectively, is formed by the oxygen shownas unbonded in the formula. Ring A is always joined to ring B in both ofFormulas I and II. One of R₁, R₂, R₅ or a hydroxyl of R₆ ring B istherefore always absent and is replaced by a glycosidic bond to ring A.

In addition, when ring C is present (m=1) and also glycosidically linkedto ring B, a second of R₁, R₂, R₅ or a hydroxyl of R₆ is absent and isreplaced by another, second glycosidic bond to ring C. When ring C ispresent and glycosidically linked to ring A, one of R₁₁, R₁₀ or ahydroxyl of R₉ is absent and replaced by a glycosidic bond to ring C.

In usual practice, R₁ and/or R₅ are replaced by the glycosidic bondswhen ring A or C are other than sialyl. However, when either of rings Aor C are sialyl, a hydroxyl of an R₆ or R₉ hydroxymethyl group isreplaced by the glycosidic bond.

The rings labeled A and B in Formula II are bonded together by theoxygen of the glycosidic bond of ring A shown to the left of the leftbracket. That oxygen atom can be bonded to one of the carbon atoms ofRing B that is shown linked to R₁, R₂, R₅ or R₆ and the appropriate R₁,R₂, R₅ or R₆ hydroxyl group is consequently absent. Thus, for example,where R₇ is hydrogen, a 1,3-; 1,2-; 1,4-; or 1,6-bond can be formedbetween rings A and B at the positions of R₁, R₂, R₅ or R₆,respectively, and the corresponding R₁, R₂, R₅ or R₆ substituent groupis absent. Where R₇ is carboxyl, that bonding can be 2,3-; 2,2-; 2,4-;or 2,6-, respectively.

Turning more specifically to Formulas I and II, it is seen thatsubstituent X of ring B can be O, S, SO, SO₂ or NR₁₆. The R₁₆ group canbe hydrogen, which is preferred, as well as a C₁ -C₁₂ acyl group, a C₁-C₁₂ alkyl group or >NR₁₆ can be a C₁ -C₁₂ alkyl N-oxide.

An R₁₆ C₁ -C₁₂ acyl group is the residuum or reaction product of acorresponding C₁ -C₁₂ carboxylic acid, and thus forms an amide with thenitrogen atom. A contemplated C₁ -C₁₂ acyl group includes formyl,acetyl, propionyl, butanoyl, iso-butanoyl, hexanoyl, octanoyl, nonanoyl,decanoyl, dodecanoyl (lauroyl ), cyclohexanecarbonyl and benzoyl.

A >NR₁₆ is a C₁ -C₁₂ alkyl N-oxide. Here, the alkyl group is as isdiscussed below and the alkylated tertiary nitrogen atom is oxidized toform the N-oxide. The symbol ">" is used to show the remaining valencesof the nitrogen that are bonded to ring carbon atoms.

A C₁ -C₁₂ alkyl group is exemplified by methyl, ethyl, propyl,iso-propyl, butyl, t-butyl, pentyl, hexyl, cyclohexyl, heptyl, octyl,nonyl, decyl and dodecyl groups.

A C₁ -C₄ acyl group as can be present as R₁, R₅ or R₁₈, or a C₁ -C₄acyloxy group of R₆ and R₉ are the acyl [RC(O)--] or acyloxy [ROC(O)--]portions of ester groups, where R is the hydrocarbon portion of the acylgroup. A C₁ -C₄ acyl group includes formyl, acetyl, propionyl, butanoyland iso-butanoyl.

A saturated alkoxide is an ether whose hydrocarbon portion is saturated.A C₁ -C₅ alkoxide can contain a length of 1 to 6 atoms in the groupwhose hydrocarbon portion can be methyl, ethyl, propyl, isopropyl, butylor pentyl groups. A 2-trimethylsilylethyl "hydrocarbon" group can bealso included. A methoxy group is preferred.

An unsaturated alkoxide is an ether like an alkoxide that furtherincludes ethylenic unsaturation in the hydrocarbon group. An unsaturatedalkoxide also can have a length up to 6 atoms in the group of which 5atoms can be carbon, and whose hydrocarbon portion includes a vinylgroup, an allyl group (prop-2-enyl), a methylvinyl group (prop-1-enyl),a 2-butenyl and a 2-pentenyl group. An allyl hydrocarbon group(allyloxy) is preferred.

An alkoxy alkoxide is an ether that includes another ether group. Analkoxy alkoxide can also have a length of 6 atoms in the group, 5 ofwhich are carbon. Exemplary 6 atom alkoxy alkoxides includemethoxylmethyoxy (--O--CH₂ --O--CH₂ --O--CH₃), ethoxylmethyoxy (--O--CH₂--O--C₂ H₅) and ethoxylethyoxy (--O--C₂ H₅ --O--C₂ H₅).

Inasmuch as saturated alkoxide, unsaturated alkoxide and alkoxy alkoxidegroups can each contain a chain length of up to 6 atoms of which 5 arecarbon, those three moieties are collectively referred to as a saturatedor unsaturated alkoxide or alkoxy alkoxide containing up to 5 carbons.

An R₁ and R₅ can also include a C₁ -C₄ alkoxycarbonyl group, whereas anR₁₆ and R₁₈ can include a C₁ -C₄ alkoxycarbonyl. The former is acarbonate, whereas the latter is a urethane. Each can be prepared byreaction of a C₁ -C₄ alkoxy (as discussed before) chloroformate with analcohol or amine, for the formation of an R₁ or R₅ group, or an R₁₆ orR₁₈ group, respectively.

R₂ can also be NR₁₇ R₁₈, where NR₁₇ R₁₈ together form a cyclic imidogroup containing 4-8 carbon atoms. Contemplated cyclic imide groupsinclude succinimido, methylsuccinimido, 2,2-dimethylsuccinimido,2,3-dimethylsuccinimido, maleimido, phthalimido, hexahydrophthalimidoand dimethylphthalimido.

An oxo group is a carbonyl group and oan be present in a B ring of theabove Formula I, II or III at the 3-position of the ring B (R₁ and R₁ ')as a ketone, or at the 1-position of ring B (R₃ and R₄) as the carbonylportion of a lactone, thiolactone or lactam.

A C₁ -C₃ alkylthio group is a thio ether in which the ether oxygen of analkoxide is replaced by a sulfur atom. The hydrocarbon portion of a C₁-C₃ alkylthio can be the same as the groups noted above for thesaturated and unsaturated alkoxide groups.

The R₂ and R₃ groups can also be absent, with their bonds forming anethylenic unsaturation between the 1- and 2-positions of the ring. Theresulting B ring saccharide unit is, thereby, a glycal.

For a particularly preferred disaccharide, R₈ is hydroxyl. An R₈ groupcan also be an N-acetamido group as where Ring A is anN-acetyl-glucosaminyl group.

Where a trisaccharide is desired, R₂ can be another saccharide that islinked to the depicted saccharide by a glycosidic bond. Exemplarysaccharides that can be so linked are noted hereinbefore.

In addition to the above-noted absence of an R₁, R₂, R₅ or R₆ group toaccount for linkage of the A and B as well as the C and B rings of theabove Formula I, two other provisos apply as to a compound of Formulas Iand II. Both of these provisos relate to compounds in which X is O, andthereby limit those compounds.

First, X is O only where one of five substituent configurations of ringB is present. Two of those configurations are oxo groups (R₁ and R₁ ')and (R₃ and R₄). The third is where either R₃ or R₄ is C₁ -C₄ alkylthio.The fourth occurs where R₁ and either R₃ or R₄ are not hydroxyl. Thefifth occurs where R₂ and R₃ are absent with their bonds formingethylenic unsaturation (ring B is a glycal) and either R₁, R₅, R₈ or R₉is not hydroxyl or R₆ is not hydroxymethyl.

Second, R₂ and R₃ are absent and replaced by ethylenic unsaturation onlywhen X is O. Thus, only a glycal is contemplated.

In a preferred embodiment, X is S, SO or SO₂ and ring B is a thiosugaror its oxygenated derivative. Where ring B is a thiosugar, preferablyR₁, R₂ and R₅ are hydroxyl, R₃ is hydrogen, hydroxyl or methoxy, R₄ ishydrogen, hydroxyl or methoxy, with the proviso that one of R₃ and R₄ ishydrogen, and R₆ is hydroxymethyl.

In yet another preferred embodiment, X is NR₁₆ and ring B forms anazasugar. Where NR₁₆ is NH, preferably R₁ is hydroxyl, R₁ ' is hydrogen,R₂ is hydroxyl or acetamido, R₃ and R₄ are both hydrogen or R₃ and R₄together form an oxo group, R₆ is hydroxymethyl and ring B is joined toring A through a glycosidic bond at R₅.

Alternatively, where X is NR₁₆, ring B can be a 1,6-dideoxy azapyranose.Where ring B is a 1,6-dideoxy azapyranose, preferably R₁ is hydrogen orhydroxyl; R₂ is hydrogen, hydroxyl, C₁ -C₅ alkoxy, halide or NR₁₇ R₁₈where R₁₇ is hydrogen or C₁ -C₄ alkyl and R₁₈ is hydrogen, C₁ -C₄ alkyl,C₁ -C₄ acyl, C₁ -C₄ alkoxycarbonyl or NR₁₇ R₁₈ together form a cyclicimido group containing 4-8 carbon atoms; R₃ and R₄ are both hydrogen; R₅is hydrogen, hydroxyl or methyl; R₆ is hydrogen or methyl with theproviso that only one of R₅ and R₆ is methyl; R₁₆ is hydrogen, C₁ -C₁₂alkyl, C₁ -C₁₂ acyl or >NR₁₆ is a C₁ -C₁₂ alkyl N-oxide; and the dideoxyazapyranose contains at least two hydroxyl groups.

Because the 1,6-dideoxy azapyranose lacks hydroxyl groups at the 1- and6-position carbon atoms, formed oligosaccharides are disaccharides withthe dideoxy azapyranose at the reducing end, which azapyranose is linkedto the saccharide at the non-reducing end (ring A) through a glycosidicbond at R₁, R₂ or R₅.

It is preferred that the oligosaccharide compounds of this inve.ntionhave a particular spatial orientation. The preferred spatial orientationfor the oligosaccharide corresponding to Formula II above is beta, andis shown below in Formula IV. ##STR6## wherein groups X, and R₁₋₉ arethe same as defined for Formula II above.

Another preferred embodiment includes a compound of Formula I in whichboth the A and C rings are joined to the B ring of the formula. Here, R₁and R₅ are both absent and are replaced by glycosyl-linked saccharides.A general structural formula for such compounds is shown as structuralFormula V, ##STR7## wherein the various R₁₋₁₅ groups present and X areas defined before.

In a preferred embodiment, ring C is an α-linked fucosyl group and ringA is a β-linked galactosyl group (Gal). In one such particularlypreferred embodiment, X is S, R₂ is hydroxyl and R₃ and R₄ are hydrogenand hydroxyl, R₆ is hydroxymethyl, and ring B has a glucoseconfiguration.

In another particularly preferred embodiment, ring C is an α-linkedfucosyl group and rings A and B are β1,4-1inked N-acetyl-glucosamines(GlcNAc)₂. In yet other embodiments, ring C is s-linked fucosyl andrings A and B are galactosylβ1,3-N-acetylglucosamine (Galβ1,3-GlcNAc) orring C is α-linked fucosyl with rings A and B beinggalactosylβ1,4-N-acetylgalactosamine (Galβ1,4-NAcGal).

The Synthetic Methods

A. Glycosyltransferase Methods

1. Procedures

Another aspect of the present invention relates to a glycosylationmethod. In accordance with the glycosylation method, an activated donormonosaccharide is admixed in an aqueous medium with an acceptorsaccharide of Formula II or III below in the presence of a catalyticamount of a glycosyltransferase having specificity for both theactivated donor monosaccharide and the acceptor saccharide to form areaction mixture: ##STR8##

wherein X, R₁, R₁ ', R₂, R₃, R₄, R₅ and R₆ (R₁₋₆) are as defined inFormula II, and the reaction mixture is maintained for a time period andunder conditions sufficient for the acceptor saccharide to beglycosylated and form a glycosylated acceptor saccharide.

As used herein, the phrase "activated donor monosaccharide" means adonor monosaccharide bonded to an activating nucleotide. Exemplary donormonosaccharides include Glc, GlcNAc, Gal, GalNAc, Man, Fuc, NeuAc andderivatives thereof such as Compounds 201-204 of Table 5 hereinafter.Activating nucleotides known in the art to have specificity for donormonosaccharides include uridine diphosphate (UDP), adenosine diphosphate(ADP), guanosine diphosphate (GDP), cytidine monophosphate (CMP) andcytidine diphosphate (CDP).

Where the donor monosaccharide is Glc, GlcNAc, Gal or GalNAc, apreferred activating nucleotide is UDP. Where the donor monosaccharideis Man or Fuc, a preferred activating nucleotide is GDP. Where the donormonosaccharide is NeuAc, a preferred activating nucleotide is CMP.Preferred activated donor monosaccharides for use in the method of thepresent invention are UDP-Gal, UDP-GalNAc, UDP-Glc, UDP-GlcNAc andCMP-NeuAc.

Activated donor monosaccharides can be obtained from commercial sources(Sigma Chem. Co., St. Louis, Mo.) or prepared from activatingnucleotides and phosphorylated monosaccharides. Activated donormonosaccharides are prepared by reacting a phosphorylated donormonosaccharide with an activating nucleotide in the presence of acatalytic amount of a nucleotide-sugar-pyrophosphorylase, an enzyme thatcatalyzes the formation of activated donor monosaccharides.

The selection of a particular nucleotide-sugar-pyrophophorylase dependsupon the nature of the phosphorylated donor monosaccharide and theactivating nucleotide used. Thus, for example, UDP-Glc pyrophophorylasecatalyzes the formation of UDP-Glc from UTP and phosphorylated Glc.Other pyrophosphorylases are well known in the art and include CMP-NeuAcsynthetase, which catalyzes the formation of CMP-NeuAc from CTP andNeuAc.

Nucleotide-sugar-pyrophosphorylases can be obtained from commercialsources, isolated from animal tissues or in recombinant form usingstandard techniques of genetic engineering, as is known.

The selection of an acceptor depends upon the desired structure of theoligosaccharide. Typically, the substituent configuration of theacceptor corresponds to the substituent nature of ring B of theoligosaccharide of Formulas I or II. The data presented below in Table 1show the correspondence between particular acceptor saccharides and thesynthesized oligosaccharide prepared using β1,4-galactosyltransferase(GalT). The reaction illustrated above in Table 1 exemplifies use ofglucosyl derivatives (Compounds 1a-z) as acceptors. Additional numberedacceptors are illustrated below the table.

                                      TABLE 1                                     __________________________________________________________________________     ##STR9##                                                                     Compound                                                                            R.sub.1    R.sub.2 R.sub.3    R.sub.4    R.sub.6'                                                                             Relative Rate           __________________________________________________________________________                                                          (%)                     1a    OH         AcNH    (H,        OH)        H      100                     1b    OH         AcNH    H          CH.sub.3 O H      75                      1c    OH         AcNH    H          CH.sub.2CHCH.sub.2 O                                                                     H      25                      1d    CH.sub.3 CHOCO.sub.2                                                                     AcNH    (H         OH)        H      0.6                     1e    OH         AcNH    (H,        OH)        CH.sub.3 CO                                                                          4                       1f    AcO        AcNH    (H,        OH)        H      0.4                     1g    CH.sub.2CHCH.sub.2 O                                                                     AcNH    H          OMe        H      0                       1h    CH.sub.3 (CH.sub.2).sub.2 O                                                              AcNH    (H,        OH)        H      0.5                     1i    H          AcNH    CH.sub.2CHCH.sub.2 O                                                                     H          H      1.0                     1j    CH.sub.2CHCH.sub.2 O                                                                     AcNH    CH.sub.3 (CH.sub.2).sub.3 O                                                              H          H      0.3                     1k    MeOCOO     AcNH    CH.sub.2CHCH.sub.2 O                                                                     H          H      a                       1l    AllylOCOO  AcNH    CH.sub.2CHCH.sub.2 O                                                                     H          H      a                       1m    MeOCH.sub.2 O                                                                            AcNH    CH.sub.2CHCH.sub.2 O                                                                     H          H      2.0                     1n    O          AcNH    CH.sub.3 O H          H      2.0                     1o    epi-OH     AcNH    (H,        OH)        H      0.04                    1p    OH         AcNH    SPh        H          H      0                       1q    O          AcNH    H          OMe        H      0.1                     1r    OH         Phthalimido                                                                           H          SPh        H      0                       1s    epi-OH     AcNH    H          OMe        H      0                       1t    OH         OH      (OH,       H)         H      100                     1u    CH.sub.2CHCH.sub.2 O                                                                     OH      (OH,       H)         H      0                       1v    MeO        OH      (OH,       H)         H      10                      1w    OH         OH      SPh        H          H      0.1                     1x    OH         OH      SPh        H          CH.sub.2 Ph                                                                          0.04                    1y    OH         H       (OH,       H)         H      60                      1z    OH         AcNH    CH.sub.3 O H          CH.sub.3                                                                             20                      3                                                     3                       4                                                     0.1                     5                                                     70                      6                                                     0.4                     D-xylose                                              90                      (GlcNAc).sub.n                                                                n = 2                                                 500                     n = 3                                                 60                      n = 4                                                 70                       ##STR10##                                                                    __________________________________________________________________________     Reaction rates for Compounds 1b-1g are given in relative terms compared t     Compound 1a (100 percent). Reaction rates for Compounds 1u6, Xylose and       GlcNAc polymers are given in relative terms compared to Compound 1t (100      percent). Parenthesized H, OH for R.sub.3 and R.sub.4 indicates that the      stereochemistry for those groups is unassigned.                               .sup.a indicates acyl migration.                                         

The preparation of acceptor saccharides varies with the nature of suchsaccharides. Deoxy-azasugars are made by a chemical-enzymatic methodbased on an aldolase-catalyzed reaction and a reductive amination.According to such a method, an azido aldehyde and a phosphate donorsubstrate are reacted in the presence of a catalytic amount of analdolase to form an azido-substituted-ketose phosphate. Theazido-substituted-ketose phosphate is then reductively cyclized byhydrogenation in the presence of a standard palladium catalyst. Thehydrogenation is carried out at greater than atmospheric pressure usinga standard hydrogenation solvent such as water, ethanol or methanol ormixtures thereof.

Alternatively, the azido-substituted-ketose phosphate isdephosphorylated prior to hydrogenation. Where such dephosphorylationoccurs, the resulting azasugar has a 1-deoxy configuration as comparedto a 1,6-dideoxy configuration that results from hydrogenation withoutprior dephosphorylation.

The substituent configuration of the azasugar is determined by theconfiguration of the azido aldehyde. Modifications on the ring nitorgenatom are typically made after reductive cyclization. For example, a C₁-C₁₂ alkyl group can be added by reductive alkylation of a correspondingaldehyde or ketone. A leaving group-substituted alkane can also be usedfor the alkylation. Exemplary leaving groups include halides,methanesulfonyl (mesyl) and p-toluenesulfonyl (tosyl) groups. Methods ofN-alkylation are well known in the art.

C₁ -C₁₂ Acyl groups can be added via an appropriate anhydride or acidhalide such as lauroyl chloride. Acylation methods are also well known.

N-Oxide derivatives are readily prepared from the N-alkyl derivatives byoxidation with hydrogen peroxide. An exemplary preparation isillustrated hereinafter.

Thiosugar acceptor saccharides such as thioglucose are available fromcommercial sources (Sigma Chemical Co., St. Louis, Mo.). Substituentconfigurations of the ring carbon atoms of such thiosugars are madeusing standard chemical techniques well known in the art. Oxidation ofthe ring S atom with H₂ O₂ to sulfoxide (SO) and sulfone (SO₂) iscarried out at room temperature. Protection of the anomeric center asmethyl thioglycoside is required to prevent ring opening.

Acceptor monosaccharides that are derivatives of naturally occurringsugars are prepared using standard chemical techniques well known in theart. Exemplary preparations of acceptor saccharides are set forthhereinafter in Example 5.

Products prepared from the reactions illustrated in connection withTable 1 can also be acceptor substrates or inhibitors for furtherglycosyl transferase reactions, as can the acceptor substrates utilizedin that table and other saccharides. Exemplary reactions and relativerates using such compounds are illustrated in Table 1a, below, wherein adisaccharide or monosaccharide reactant compound (acceptor substrate)has the same number as an acceptor or product compound of Table 1[Compounds 1a, 1t, 2a, 2i, 2t, 3, 5, 6 and (GlcNAc)₂ ] or a compounddiscussed elsewhere herein (Compound 7, 8 and 10a), and the transferaseutilized was fucosyl α1,3/1,4 transferase (FucT; EC 2.4.1.65). Reactionssuch as those shown in Table 1a thus utilize compounds such as thoseshown by structural Formulas III and IV to prepare compounds such asthose of structural Formulas I, II and V. An exemplary branchedtrisaccharide-forming reaction is shown above the table. The data ofTable la are also reported in Dumas et al., BioMed. Chem. Lett.,1:425-428 (1991), as are other data.

                                      TABLE 1a                                    __________________________________________________________________________     ##STR11##                                                                    Compound*    R.sub.1                                                                             R.sub.2 R.sub.3                                                                            R.sub.4      X     Relative Rate              __________________________________________________________________________                                                       (%)                        2a           OH    AcNH    (H   OH)          O     100                        2i           H     AcNH    (H   CH.sub.2CHCH.sub.2 O)                                                                      O     IC.sub.50 > 124 mM         7            OH    OH      (H   OH)          S     310                        10a          OH    OH      (H   H)           NH    IC.sub.50 = 40mM           2t           OH    OH      (H   OH)          O     120                        8                                                  IC.sub.50 > 125 mM         (GlcNAc).sub.2                                     23                         Galβ1,3GlcNAc                                 580                        Galβ1,4GalNAc                                 27                         1t                                                 5                          1a                                                 3                          5                                                  7                          3                                                  5                          6                                                  7                          __________________________________________________________________________     *Reactant compound (acceptor substrate)                                  

Glycosyltransferase used in the glycosylation method has specificityboth for the activated donor monosaccharide and the acceptor saccharide.That is, the glycosyltransferase is capable of transferring theactivated donor monosaccharide to the acceptor saccharide and forming aglycosidically-linked oligosaccharide of a predetermined configuration.

Exemplary glycosyltransferases include those enzymes that catalyze theformation of the products in Table 2, below. See, also, Beyer et al.,Adv. Enzymol., 52:23-161 (1981). Further, as exemplified hereinafter,glycosyltransferases can utilize non-naturally occurringoligosaccharides.

                  TABLE 2                                                         ______________________________________                                        I.         Sialyltransferase                                                             Siaα2,6Gal                                                              Siaα2,3Gal                                                              Siaα2,6GalNAc                                                           Siaα2,6GlcNAc                                                           Siaα2,8Sia                                                              Siaα2,4Gal                                                              Siaα2,4GlcNAc                                                           Siaα2,6Man                                                   II.        Fucosyltransferase                                                            Fucα1,2Galβ                                                        Fucα1,4GlcNAcβ                                                     Fucα1,3GlcNAcβ                                                     Fucα1,3Glc                                                              Fucα1,6GlcNAcβ                                                     Fucα1,6Galβ                                                        Fucα1,3Galβ                                                        Fucα1,3Fuc                                                   III.       Galactosyltransferase                                                         Galβ1,4Glc                                                               Galβ1,4GlcNAc                                                            Galβ1,3GlcNAc                                                            Galβ1,3diglyceride                                                       Galβ1,6GlcNAc                                                            Galβ1,3GalNAc                                                            Galβ1,6GalNAc                                                            Galα1,3GalNAc                                                           Galα1,3Gal                                                              Galα1,4Gal                                                              Galβ1,4Gal                                                               Galβ1,6Gal                                                               Galβ1,4Xyl                                                    IV.        N-Acetylgalactosaminyltransferase                                             GalNAcα1,3Galβ                                                     GalNAcβ1,4Gal                                                            Iduronic Acid                                                                 GalNAcβ1,3Gal                                                            GalNAcα1,3GalNAc                                                        (GalNAcβ1,4GluUAβ1,3).sub.n                                         (GalNAcβ1,4IdUAα1,3).sub.n                              V.         N-Acetylglucosaminyltransferase                                               GlcNAcβ1,4GlcNAc                                                         GlcNAcβ1,2Man                                                            GlcNAcβ1,4Man                                                            GlcNAcβ1,6Man                                                            GlcNAcβ1,3Man                                                            GlcNAcβ1,3Gal                                                            GlcNAcβ1,4Gal                                                            GlcNAcβ1,6Gal                                                            GlcNAcα1,4Gal                                                           GlcNAcα1,4GlcNAc                                                        GlcNAcβ1,6GalNAc                                                         GlcNAcβ1,3GalNAc                                                         GlcNAcβ1,4GlcUA                                                          GlcNAcα1,4GlcUA                                                         GlcNAcα1,4IdUA                                               ______________________________________                                    

Glycosyltransferases can be obtained from commercial-sources (SigmaChem. Co., St. Louis, Mo.; Boehringer Mannheim, Indianapolis, Ind. andGenzyme, Cambridge, Mass.), isolated and purified from microbial, plantor animal tissues or in recombinant form using well known techniques ofgenetic engineering.

As used herein, the phrase "catalytic amount" means that amount of anenzyme at least sufficient to catalyze, in a non-rate limiting manner,the conversion of that enzyme's substrate to product.

The catalytic amount of a particular enzyme varies according to theconcentration of that enzyme's substrate as well as to reactionconditions such as temperature, time and pH value. Means for determiningthe catalytic amount for a given enzyme under preselected substrateconcentrations and reaction conditions are well known to those of skillin the art.

Admixing comprises mixing each ingredient with each of the otheringredients in a suitable aqueous medium (solvent) to form a reactionmixture. The reaction mixture is maintained under biological reactionconditions of temperature, pH, solvent osmolality, ionic composition andambient atmosphere for a period of time sufficient to glycosylate theacceptor saccharide and form a glycosylated acceptor saccharide.

Temperature can range from about 15° C. to about 40° C. Preferablytemperature is from about 20° C. to about 40° C. and, more preferablyfrom about 25° C. to about 37° C.

The pH value can range from about 6.0 to about 11.0. Preferably, the pHvalue is from about 6.5 to about 8.5 and, more preferably about 7.0 toabout 7.5. The pH value is maintained by buffers in the aqueous solvent.The buffer is devoid of chelators that bind enzyme cofactors such asMg⁺² or Mn⁺². The selection of a buffer is based on the ability of thebuffer to maintain pH value at the desired level. Where the pH value isabout 7.5, a preferred buffer is HEPES.

The osmolality and ionic composition of the aqueous solvent are designedand selected to solubilize the ingredients of the reaction mixture andto provide cofactors for the enzymes contained in the reaction mixture.The osmolality of the aqueous solvent including the buffer is preferablyfrom about 100 mOsm to about 300 mOsm.

The reaction time and conditions for the synthesis of an oligosaccharidevary with the nature of the monosaccharide acceptor. Where themonosaccharide derivative is Compound 5 from Table 1, the reaction timeis about 48 hours and the reaction occurs in a buffered aqueous solutionat a temperature of about 37° C. (Example 1A). When the monosaccharideacceptor is Compound 1e or 1i from Table 1, the reaction time is about96 hours at the same temperature (Examples 1B and 1C).

Under certain circumstances, when the monosaccharide acceptor has ahydrogen or a hydroxyl group in an s-orientation at the carbon atom atposition 2 (i.e., R₂ is hydrogen or hydroxyl in Formula III), thereaction conditions include lactalbumin and, preferably α-lactalbumin.

It is noted that the synthetic method of the present invention does notprovide any 3-O-acyl oligosaccharides prepared with GalT. When 3-O-acylmonosaccharide acceptors are used, the formed product is a 6-O-acylatedoligosaccharide. For example, when 3-O-acetyl-N-acetylglucosamine wasused as the monosaccharide acceptor, Compound 2e from Table 1 wasobtained indicating a migration of the acetyl group to position 6 of theN-acetylglucosamine moiety. No other byproduct was obtained.

In addition to the acetyl group, methoxycarbonyl, chloroacetyl, andallyloxycarbonyl groups also showed 3→6 O-acyl migration. The half-lifefor each of these migrations was about three hours for acetyl,methoxycarbonyl, and allyloxycarbonyl group, and less than three hoursfor the chloroacetyl group at room temperature and pH 7.0 as measured byNMR.

The synthesis of 3O-acyl-GlcNAc is straightforward. Starting from thereadily available 4,6-O-benzylidene derivative, various acyl group canbe introduced to the 3-O position.

To further study this unexpected acyl migration,3-O-acetyl-N-acetylglucosamine was incubated at pH 7.0 in the absence ofthe GalT enzyme and a ¹ H-NMR spectrum was taken. It was observed thatthe intensity of a new peak at 1.82 ppm increased while the signal at1.90 ppm (C H₃ CONH--) decreased, and a downfield shift of H-6 andupfield shift of H-3 were observed at the same time. After 24 hours, 90percent of the original compound was converted to the new product whichwas identical to an authentic 6-O-methoxycarbonyl-N-acetylglucosamineprepared separately. The identity was further confirmed by thehigh-resolution mass spectroscopy analysis.

6-O-Acetyl-N-acetylglucosamine was then studied as a substrate for GalTand it was found to be about 10 times as effective as the 3-O-acylisomer under the same conditions. A separate synthesis of the 6-O-acetyldisaccharide was then accomplished in 70 percent isolated yield with theuse of 6-O-acetyl-N-acetylglucosamine as a substrate. It is worth notingthat 6-O-acetyl-N-acetylglucosamine was easily prepared in 82 percentyield from GlcNAc and isopropenyl acetate in anhydrous dimethylformamidecatalyzed by subtilisin.

The reaction rate and yield of the glycosylation method can be enhancedby providing for the in situ regeneration of activated donormonosaccharide.

In a preferred embodiment, the glycosylation method comprises the stepsof:

(a) admixing in the presence of each other in an aqueous medium

(i) an acceptor saccharide;

(ii) a donor monosaccharide;

(iii) an activating nucleotide having specificity for the donormonosaccharide;

(iv) an activated donor monosaccharide regenerating system;

(v) a pyrophosphate scavenger; and

(vi) catalytic amounts of a glycosyltransferase having specificity forboth the activated form of the donor monosaccharide and the acceptorsaccharide and a nucleotide-sugar-pyrophosphorylase having specificityfor both the donor monosaccharide and the activating nucleotide to forma reaction mixture; and

(b) maintaining the reaction mixture for a time period and underconditions sufficient for the acceptor saccharide to be glycosylated andform a glycosylated acceptor saccharide.

The donor monosaccharides, activating nucleotides, glycosyltransferasesand nucleotide-sugar-pyrophosphorylases used in this preferred methodare the same as set forth above.

The activated donor monosaccharide regenerating system comprises aphosphate donor and a catalytic amount of a kinase that catalyzes thetransfer of phosphate from the phosphate donor to an activatingnucleotide.

The phosphate donor of the regenerating system is a phosphorylatedcompound, the phosphate group of which can be used to phosphorylate anucleoside diphosphate such as ADP or CDP. The only limitation on theselection of a phosphate donor is that neither the phosphorylated northe dephosphorylated forms of the phosphate donor can substantiallyinterfere with any of the reactions involved in the formation of theglycosylated acceptor saccharide. Preferred phosphate donors arephosphoenolpyruvate (PEP) and acetyl phosphate. A particularly preferredphosphate donor is PEP.

The selection of a particular kinase for use in accordance with thepresent invention depends upon the phosphate donor employed. When acetylphosphate is used as the phosphate donor, the kinase is acetyl kinase.When PEP is used as the phosphate donor, the kinase is pyruvate kinase.Other kinases can be employed with other phosphate donors as is wellknown to those of skill in the art. Kinases are commercially available(Sigma Chem. Co., St. Louis, Mo.; Boehringer Mannheim, Indianapolis,Ind.).

As used herein, the term "pyrophosphate scavenger" refers to substancesthat serve to remove inorganic pyrophosphate from a reaction mixture ofthe present invention. Inorganic pyrophosphate (PPi) is a byproduct ofsome activated donor monosaccharides.

Produced PPi can feed back to inhibit other enzymes such thatglycosylation is reduced. However, PPi can be removed by metabolic meanssuch as catabolism or by physical means such as sequestration by a PPibinding substance. Preferably, PPi is removed by metabolic means usinginorganic pyrophosphatase, a commercially available PPi catabolic enzyme(Sigma Chem. Co., St. Louis, Mo.; Boehringer Mannheim, Indianapolis,Ind.), and that or a similar enzyme serves as the pyrophosphatescavenger.

The acceptor saccharide used in the preferred glycosylation method canbe an acceptor monosaccharide, such as defined by structural FormulaIII, a naturally occurring mono- or oligosaccharide, or anoligosaccharide having structural Formula II, or more specifically,structural Formula IV.

By way of example, an oligosaccharide, such as a sialylated glycosylcompound, can be synthesized with the in situ regeneration of CMP-sialicacid according to Scheme 1 below, that is also referred to as cycle Ahereinafter. Such a synthesis results in a linear oligosaccharide inwhich ring C of structural Formula I is linked to ring A, and ring A islinked to ring B.

A compound such as one of Compounds 201-204 that are illustratedhereinafter in Table 5 can be utilized in place of NeuAc. Use of suchcompounds provides compounds of structural Formulas I, II and IV having3-substituted-1,2-dihydroxypropyl groups. ##STR12##

According to Scheme 1, CMP is converted to CDP catalyzed by nucleosidemonophosphate kinase (NMK) in the presence of ATP, which is regeneratedfrom its byproduct ADP catalyzed by pyruvate kinase (PK) in the presenceof phosphoenolypyruvate (PEP). CDP is further converted to CTP with PEPcatalyzed by PK. CTP reacts with NeuAc catalyzed by CMP-NeuAc synthetaseto produce CMP-NeuAc. The byproduct inorganic pyrophosphate is scavengedby pyrophosphatase (PPase). Sialylation of Galβ1,4GlcNAc is accomplishedby CMP-NeuAc and Siaα2,6Gal sialyltransferase. The released CMP is againconverted to CDP, to CTP and to CMP-NeuAc.

In accordance with such a method, therefore, there are reacted: a sialicacid such as N-acetylneuraminic acid (NeuAc) as the donormonosaccharide; a Galβ1,4GlcNAc (N-acetyllactosamine; LacNAc) as theacceptor disaccharide; a CMP-sialic acid regenerating system as theactivated donor monosaccharide regenerating system; a pyrophosphatescavenger such as inorganic pyrophosphotase, and catalytic amounts of aCMP-sialic acid synthetase as the nucleotide-sugar-pyrophosphorylase anda sialyltransferase as the glycosyltransferase having substratespecificity for the acceptor saccharide.

As used herein, the term "a sialic acid" means neuraminic acid(5-amino-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonic acid), andderivatives such as an N- or O-acetyl derivative of neuraminic acid.Preferably, the sialic acid is an N-acetyl derivative of neuraminic acid(NeuAc), which has been reported as a naturally occurring sialic acid invarious animal species. Schauer, Adv. Carbohydr. Chem. Biochem., 40:131(1982).

A sialic acid derivative can be substituted at the carbon atom atpositions 4, 5, 7, 8 or 9 of the sialic acid as herein defined.Exemplary derivatives at the above positions include a fluoro or deoxygroup at positions 5, 7, 8 or 9, an C₁ -C₃ acyl or amino acyl of anamino from an amino acid, and phosphoryl. Positions 5 or 9 can also besubstituted with an azido group. Particularly preferred sialic acids areNeuAc, N-lactylneuraminic acid, 9-0-acetyl-NeuAc,9-deoxy-9-fluoro-NeuAc, and 9-azido-9-deoxy-NeuAc. A sialic acid used inaccordance with the present invention can be obtained commercially(Sigma Chemical Company, St. Louis, Mo.) or isolated from various animaltissues. Schauer et al., Biochem. Soc. Symp., 40:87 (1974).

As used herein, the term "a glycosyl compound" refers to an organiccompound having one or more glycosyl residues. Preferred glycosylresidues are Gal, GlcNAc, GalNAc, NeuAcGalβ1,4GlcNAc. The glycosylresidue acts as the acceptor for the sialic acid, and therefore musthave an appropriate hydroxyl group available to accept the sialic acidgroup.

The CMP-sialic acid regenerating system used in the present inventioncomprises cytidine monophosphate (CMP), a nucleoside triphosphate, aphosphate donor, a kinase capable of transferring phosphate from thephosphate donor to nucleoside diphosphates and a nucleosidemonophosphate kinase capable of transferring the terminal phosphate froma nucleoside triphosphate to CMP.

Nucleoside triphosphates suitable for use in accordance with theCMP-sialic acid regenerating system are adenosine triphosphate (ATP),cytidine triphosphate (CTP), uridine triphosphate (UTP), guanosinetriphosphate (GTP), inosine triphosphate (ITP) and thymidinetriphosphate (TTP). A preferred nucleoside triphosphate is ATP.

Nucleoside monophosphate kinases are enzymes that catalyze thephosphorylation of nucleoside monophosphates. Nucleoside monophosphatekinase (NMK) and myokinase (MK) used in accordance with the CMP-sialicacid regenerating system of the present invention are used to catalyzethe phosphorylation of CMP. NMK's are commercially available (SigmaChem. Co., St. Louis, Mo.; Boehringer Mannheim, Indianapolis, Ind.).

Because of the self-contained and cyclic character of this glycosylationmethod, once all the reactants and enzymes are present, the reactioncontinues until the first of the phosphate donor, donor monosaccharideor acceptor saccharide is consumed.

Thus, in the sialylation example, CMP is converted to CDP, whoseconversion is catalyzed by nucleoside monophosphate kinase in thepresence of added ATP. ATP is catalytically regenerated from itsbyproduct, ADP, by pyruvate kinase (PK) in the presence of addedphosphoenolpyruvate (PEP). CDP is further converted to CTP, whichconversion is catalyzed by PK in the presence of PEP. CTP reacts withsialic acid to form PPi and CMP-sialic acid, the latter reaction beingcatalyzed by CMP-sialic acid synthetase. Following sialylation of theglycosyl compound, the released CMP re-enters the regenerating system toreform CDP, CTP and CMP-sialic acid. The formed PPi is scavenged asdiscussed before, and forms inorganic phosphate (Pi) as a byproduct.Pyruvate (PYR) is also a byproduct.

The concentration or amount of the various reactants used in theglycosylation method of the present invention depend upon numerousfactors including reaction conditions such as temperature and pH value,and the amount of acceptor saccharide to be glycosylated. Because theglycosylation method of the present invention permits regeneration ofactivating nucleotides and activated donor monosaccharides andscavenging of produced PPi in the presence of catalytic amounts of theenzymes, the method is limited by the concentrations or amounts of donormonosaccharide, phosphate donor and acceptor saccharide. The upper limitfor the concentrations of reactants that can be used in accordance withthe method of the present invention is determined by the solubility ofsuch reactants.

In a preferred embodiment, glycosylation is limited by the concentrationof donor monosaccharide. According to such an embodiment, theconcentrations of activating nucleotides, phosphate donor, acceptorsaccharide and enzymes are selected such that glycosylation proceedsuntil the donor monosaccharide is consumed.

By way of example, when the concentration of sialic acid is about 20 mM,preferred concentrations of the other non-enzyme reactants are about 20mM for the glycosyl compound, about 20-200 μM for CMP, about 2-20 μM forthe nucleoside triphosphate and about 40 mM for the phosphate donor.Thus, the ratio of the concentration of these reactants to theConcentration of sialic acid is preferrably about 0.01-0.1:1 for theglycosyl compound, about 0,001-0.01:1 for CMP, about 0,001-0.01:1 forthe nucleoside triphosphate and about 2:1 for the phosphate donor.

The glycosylation method further comprises isolating the glycosylatedacceptor saccharide. Isolation comprises recovering the glycosylatedacceptor saccharide from the reaction mixture. Means for recovering theglycosylated acceptor saccharide include gel filtration, columnchromatography, paper chromatography, affinity chromatography,extraction, precipitation and the like.

In a preferred embodiment, isolation is accomplished by lyophilizing thereaction mixture to reduce the volume of the reaction mixture, applyingthe lyophilized reaction mixture to a gel filtration column of about200-400 mesh and eluting the sialylated glycosyl compound from thefiltration column. Where such an embodiment is used to isolatesialylated glycosyl compounds, such compounds can be recovered with ayield of about 97 percent (see Example 2).

Where the acceptor saccharide is an acceptor oligosaccharide, such anacceptor oligosaccharide can itself be prepared in the reaction mixtureof the glycosylation method. In such an embodiment, the reaction mixturefuther includes:

(a) a second acceptor saccharide;

(b) a second donor monosaccharide;

(c) a second activating nucleotide that has specificity for the seconddonor monosaccharide;

(d) a second activated donor monosaccharide regenerating system; and

(e) catalytic amounts of (i) a second glycosylatransferase havingspecificity for both the activated form of the second donormonosaccharide and the second acceptor saccharide and (ii) a secondnucleotide-sugar-pyrophosphorylase having specificity for both thesecond donor monosaccaride and the second activating nucleotide.

The second acceptor saccharide can also be an acceptor oligosaccharide.The second glycosyltransferase is pareferably selected from the group ofenzymes set forth above in Table 2.

The second donor monosaccharide, second activating nucleotide, secondglycosyltransferase and second nucleotide-sugar-pyrophosphorylase can bethe same as or different from the donor monosaccharide, activatingnucleotide, glycosyltransferase and nucleotide-sugar-pyrophosphorylaseset forth above.

Further, the second activated donor monosaccharide regeneration systemcan be wholly or in part identical to the activated donor monosaccharideregeneration system set forth above.

By way of example, the acceptor saccharide (Galβ1,4GlcNAc; LacNAc) ofthe sialylation method of Scheme 1 above can be prepared in the samereaction mixture as illustrated below in Scheme 2, wherein dotted linesand the letters A, B and C are used to separate and identify the threereaction cycles that take place within a single aqueous reaction mixturein a single vessel. This synthesis also results in formation of a linearoligosaccharide in which ring A is linked to each of rings B and C.##STR13##

The preparation of LacNAc by cycle C in Scheme 2 is combined with theabove-mentioned sialyation method (A+B in Scheme 2).

As indicated in Scheme 2, the components of cycle C included in thereaction mixture are a-second donor monosaccharide (Gal), a secondacceptor saccharide (GlcNAc), a second activating nucleotide (UDP), asecond activated donor monosaccharide regenerating system (a phosphatedonor-PEP; a Kinase-PK), which is identical to part of the activateddonor monosaccharide regenerating system of Scheme 1 (cycle A), a secondglycosyltransferase (β1,4galactosyltransferase) and a secondnucleotide-sugar-pyrophosphorylase (UDP-Glc pyrophosphorylase).

In Scheme 2, the activated second donor monosaccharide (UDP-Gal) isprepared from the epimerase-catalyzed conversion of UDP-Glc, which is inturn synthesized from UTP and glucose 1-phosphate (Glc-1-P). Such amodification of the generation of the activated galactose (UDP-Gal)represents a still further embodiment of the glycosylation method.

A still further embodiment comprises the generation of sialic acid(NeuAc) from ManNAc (cycle B, Scheme 2).

An enzymatic aldol reaction (B in Scheme 2) is first introduced to theScheme 1: ManNAc is converted to NeuAc catalyzed by NeuAc aldolase (EC4.1.3.3) in the presence of pyruvic acid. Although NeuAc aldolase alsocatalyzes the reverse reaction (NeuAc to ManNAc and pyruvate), theproduced NeuAc is irreversibly incorporated into cycle A via CMP-NeuAccatalyzed by CMP-sialic acid synthetase coupled with inorganicpyrophosphatase (PPase)-catalyzed decomposition of the releasedinorganic pyrophosphate. The sialyl LacNAc is obtained after a Bio-GelP-2 column chromatography.

Details of the method illustrated in Scheme 2 are provided in Example 3.

The glycosylated acceptor saccharide can, in turn, serve as an acceptorsaccharide for additional glycosylation reactions. For example, thesialyl LacNAc produced in accordance with Scheme 2 can be-furtherglycosylated as illustrated below in Scheme 3 to form sialyl Le^(x),wherein dotted lines and capatalized A-D are as described previously.This synthesis results in a branched oligosaccharide in which each ofrings A and C is linked to ring B, which here is a disaccharide.##STR14##

According to such a further glycosylation (cycle D, Scheme 3), sialylLacNAc serves as an acceptor saccharide in the reaction mixture ofScheme 2, further comprising α1,3 fucosyltransferase, fucose 1-phosphate(Fuc-1-P), GTP and GDP-Fuc pyrophosphorylase.

All of the above reactions, as exemplified in Scheme 3, can proceed inthe presence of one another (i.e., the same reaction vessel) because ofthe unique specificity of the glycosyltransferase and pyrophosphorylaseenzymes employed. For example, Gal is not transferred to any otheracceptor saccharide than GlcNAc because the β1,4 galactosyltransferaseenzyme used does not have specificity for such other acceptorsaccharides. Thus, a unique oligosaccharide can be designed andsynthesized in accordance with the glycosylation method of the presentinvention by selecting and using specific enzymes.

2. Components

(a) Acceptor Saccharides

As set forth above, an acceptor saccharide can be an aza sugar, such asa deoxy-aza sugar. The synthesis of several dideoxy-azasugars and theirderivatives began with the aldol condensation of(RS)3-azido-2-hydroxypropanal and DHAP catalyzed by FDP aldolase,rhamnulose-1-phosphate aldolase, or fuculose-1-phosphate aldolase, or(RS)3-azido-2-hydroxypropanal plus acetaldehyde, acetone, orpropionaldehyde, in the case of DERA. (RS)3-Azido-2-hydroxypropanal isprepared by the acid hydrolysis of 3-azido-2-hydroxypropanal diethylacetal as described in Durrwachter et al., J. Org. Chem., 53:4175(1988). The addition of one of the above-mentioned aldolases to(RS)3-azido-2-hydroxypropanal plus DHAP provided Compounds 101, 104 or108 as are shown on the left side of Scheme 4, below. Bothrhamnulose-1-phosphate aldolase and fuculose-1-phosphate aldolase acceptthe (S)-aldehyde as substrate, whereas FDP-aldolase is selective for the(R)-enantiomer [Pederson et al., Tetrahedron Lett., 29; 645(1988)].

Palladium (Pd)-mediated reductive amination of phosphorylated Compounds104 and 108 gave Compounds 106, 109 and 110 (at about a 1:1 ratio)respectively, each in approximately 90 percent total yield. In the samemanner, the phosphorylated Compound 101c was hydrogenated directly toCompound 103c, also in high yield. These products are shown on the rightside of Scheme 4. ##STR15##

The synthesis of N-acetyl derivatives of an azasugar of Scheme 4proceeds similarly from the reaction of DHAP with(RS)3-azido-2-acetamidopropanal diethyl acetal, which is prepared from3-azido-2-hydroxypropanal as described in Pederson et al, J. Org. Chem.,55:4897 (1990). A key element in the synthesis of these N-acetyldideoxy-azasugars is the preparation of compound. IV and its enantiomeras is shown in Scheme 5, below, and in which Roman numerals are used forintermediate compounds leading to the azido α-ketose phosphate.##STR16##

Thus, Compound I (>98 percent ee) prepared previously [Von der Osten etal., J. Am. Chem. Soc., 111:3924 (1989); Pederson et al., Heterocycles,28:477 (1989)]was converted-to Compound II [Pederson-et al., J. Org.Chem., 55:489 (1990)] in step a, followed by N-acetylation to CompoundIIIa (95 percent ee). Nucleophilic opening of aziridine Compound IIIawith sodium azide in the presence of zinc chloride (ZnCl₂) gave CompoundIVa in 60 percent yield, in step c. The other protecting "R" groups areas shown in Scheme 5. Higher yields (75-86 percent) were obtained withother protecting groups (e.g. IIIb-IIId; Cbz=carbobenzoxy,Ts=p-toluenesulfonyl). The protecting group of IIId can be removed byfluoride [Weinreb et al., Tetrahedron Lett., 2099 (1986)].

Acid hydrolysis was used to unmask the aldehyde protecting group ofCompound IVa. The unmasked product of Compound IVa (3 equivalents) wascondensed with 1 equivalent of dihydroxyacetone phosphate (DHAP) in thepresence of FDP aldolase at pH 6.5 to give Compound 101b (60 percent) instep d. Palladium catalyzed cyclization of Compound 101b providedCompound 103b, step e.

Compound 101d was prepared similarly from the enantiomer of Compound I.Thus, starting with racemic Compound I, a mixture of Compounds 103b and103d in a 12:1 ratio was obtained.

Starting with enantiomerically pure aldehyde substrates, Compounds 103band 103d were obtained separately.

The reductive aminations are all diastereoselective and consistent withthe previous finding [von der Osten et al. J. Am. Chem. Soc., 111:3924(1989)]that hydrogen would attack the imine intermediate in a facialselective manner to avoid the torsional strain developed during thereduction (e.g., reactions with Compounds 101a-101f and Compound 104).An additional finding in this study is that hydrogen always approachesfrom the side opposite to the-axial substituent (e.g., reactions withCompounds 101a, 101b, 101e, 104 and 108) and this steric effect seems tooverride the torsional strain effect. The A₁,2 strain (e.g., compounds101 or 104) seems not to affect the stereochemical course of thereduction. 10 compound 103c was N-methylated to give Compound 117.Similar alkylation with a longer alkyl group such as lauryl or butylprovides Compounds 120 and 121, respectively. ##STR17##

N-oxidation of Compound 117 with hydrogen peroxide (H₂ O₂) resulted in asingle stereoisomer with the N-methyl (CH₃) group at the equatorialposition, as indicated in Compound 118. Assignment of thestereochemistry was based on a strong nuclear Overhauser effect (NOE)observed between the various groups of a model compound. ##STR18##

Synthesis of Compounds 114a-c also begins with3-azido-2-hydroxypropanal, with formation of the precursor azaketosebeing catalyzed by DERA. DERA is unique in that it can catalyze thealdol condensation of two aldehydes. Therefore, in the case of Compound114a the reactants were (RS)3-azido-2-hydroxypropanal and acetaldehydeto provide Compound 113a; Compound 114b was formed via the reaction of(RS)3-azido-2-hydroxypropanal and acetone to form Compound 113b; andCompound 114c was formed by reacting (RS)-3-azido-2-hydroxypropanal withpropionaldehyde to form Compound 113c. None of the resultingazidoketoses or azidoaldoses contained phosphate groups, so reductivecyclization yielded Compounds 114a-c directly from parent CompoundsCompounds 113a-c and 114a-c are shown below. ##STR19## (b) Enzymes (i)Nucleotide-sugar-pyrophosphorylase

As set forth above, nucleotide-sugar-pyrophosphorylases can be obtainedfrom commercial sources, isolated and purified from tissue or obtainedin recombinant form.

For example, CMP-sialic acid synthetase can be isolated and purifiedfrom cells and tissues containing the synthetase enzyme by procedureswell known in the art. See, e.g., Gross et al., Eur. J. Biochem.,168:595 (1987); Vijay et al., J. Biol. Chem., 250(1):164 (1975); Zapataet al., J. Biol. Chem., 264(25):14769 (1989) and Higa et al., J. Biol.Chem., 260(15) :8838 (1985). The gene for this enzyme has also beensequenced. Vann et al., J. Biol. Chem., 262:17556 (1987). Shames et al.also recently reported overexpression of the gene for use in a gramscale synthesis of CMP-NeuAc. Glycobiology, 1:187 (1991).

In one embodiment, grey matter from bovine brain is homogenized, thehomogenate centrifuged to form a pellet and a supernatant, and thesupernatant lyophilized to yield a powder. The lyophilized powder isreconstituted in distilled water, homogenized and centrifuged to yield asupernatant and a fluffy pellet containing the CMP-sialic acidsynthetase. The synthetase enzyme is double extracted from the fluffypellet with KCl to yield a semi-purified extract. Contaminating amountsof nucleoside phosphatase and O-acetyl esterase are removed from thesemi-purified extract by sequential ammonium sulfate precipitation anddialysis. Higa et al., J. Biol. Chem., 260(15):8838 (1985).

In another embodiment, CMP-sialic synthetase is obtained fromtransformed host cells using genetic engineering and recombinant DNAtechnologies. One such method for obtaining CMP-sialic acid synthetasehas been reported by Zapata et al., J. Biol. Chem., 264(25):14769(1989). In this embodiment, plasmid pSR₃₅ containing the gene for E.coli native CMP-sialic acid synthetase is digested with Eco RI and HindIII to yield a 2.7 kb fragment, which is inserted into Eco RI-Hind IIIdigested vector pKK223-3 (Pharmacia LKB Biotechnology Inc.) to formplasmid pWA1. Plasmid pWA1 is then used to transform E. coli. Thetransformed E. coli are reported to express native CMP-sialic synthetaseto a level 10-30 fold higher than in non-transformed bacteria. Zapata etal., J. Biol. Chem., 264(25):14769 (1989).

In another and preferred embodiment, a native or modified CMP-sialicacid synthetase is obtained from host cells transformed with a novelbacteriophage lambda vector system recently described by Huse et al.,Science, 246:1275 (1989). Detailed descriptions of this method forobtaining native or modified CMP-sialic acid synthetase are set forth inExamples 2 and 9.

According to one aspect of this preferred embodiment, genomic DNA isextracted from E. coli strain K235 (ATCC 13207), and the gene forCMP-sialic acid synthetase is isolated via polymerase chain reaction(PCR) amplification in the presence of two custom-designedpolynucleotide primers (see Example 2). One primer contains an Eco RIrestriction site, a ribosomal binding sequence, a start codon, and anoligonucleotide corresponding to the N-terminal hexapeptide of theenzyme The second primer contains, from 3' to 5' an Xba I restrictionsite, a stop codon, a decapeptide tag sequence, and a sequencecorresponding to the C-terminal heptapeptide of the enzyme. Theamplified gene was cloned into a lambda ZAP™ (Stratagene CloningSystems, La Jolla, Calif.) vector at the Eco RI and Xba I sites for theconstruction of a phagemid (CMPSIL-1) for expression in E. coli of amodified synthetase enzyme that includes the decapeptide tag.

The decapeptide tag serves as a marker to facilitate the selection ofpositive clones and can be removed by another PCR (polymerase chainreaction) with primers that do not contain the decapeptide tag sequenceif the native enzyme is desired.

The native enzyme was also prepared by expression from another phagemid(CMPSIL-W10) in E. coli. This phagemid was constructed from CMPSIL-1 asdiscussed hereinafter by removal of the decapeptide tag condons usingPCR technology, followed by digestion of CMPSIL-1 and the PCR productwith Eco RI and Xba I, and religation of the appropriate DNA sequences.

The native and the modified enzymes have similar k_(cat) and K_(m) forNeuAc and CTP. The modified enzyme is more active than the native enzymeat higher pH values (See Example 9 hereinafter). Studies on specificityindicate that both enzymes have high specific activity for C-9 modifiedNeuAc derivatives at neutral pH (See Example 9 hereinafter).

It was particularly surprizing that DNA coding for an enzyme as large asnative or modified CMP-sialic acid synthetase could be successfullycloned into and translated from this phage vector. Prior reports hadonly described use of DNA coding for an antibody Fab fragment (about50,000 kd), which is about 15-20 percent of the size of the present DNAand protein.

Transfected E. coli containing phagemid CMPSIL-1 produce approximately100 U/L of CMP-sialic acid synthetase as the modified enzyme compared to<0.1 U/L for the wild-type, non-transformed strain, corresponding toa >1,000-fold increase of enzyme activity. Such a transformed E. coliwas deposited on February 19, 1991 with the American Type CultureCollection, Rockville, Md. and assigned ATCC accession No 68531. E. colitransformed with phagemid CMPSIL-W10 produce approximately 35 U/L ofnative CMP-sialic acid synthetase.

(ii) Glycosyltransferases

Glycosyltransferases can also be obtained from a variety of sources. Byway of example, β1,4 galactosyltransferase from bovine milk(GalT)-can-be produced in recombinant form. GalT, like many otherglycosylransferases, is primarily present in the golgi apparatus in amembrane-bound form, which, after proteolysis generates a soluble activeform, the so-called "catalytic domain" which soluble form appears inbody fluids such as milk and serum. Paulson, et al., J. Biol. Chem.,264:17615 (1989).

The catalytic domain of bovine GalT is composed of 324 amino acidscorresponding to the C-terminal sequence of the intact 402-amino acidmembrane-bound enzyme. The catalytic or active domain of human GalT isvery similar to bovine GalT both in sequence (>90 percent homology) andsubstrate specificity. The active domain has been cloned into an E. coliexpression system pIN-GT (See FIG. 1). This expression systemincorporates a fusion of the catalytic domain of GalT with the signalsequence of omp A, the major lipoprotein of prokaryotes, so that theenzyme is translocated to the periplasmic space where it is releasedfrom the signal sequence by action of signal peptidase to giveenzymatically active GalT. Aoki, et al., EMBO, 9:3171 (1990).

The recombinant GalT enzyme produced via this expression system containsan additional tripeptide Ala-Glu-Leu attached to the N-terminal Thrresidue of the soluble GalT. To improve the expression level, plasmidpIN-GT in E. coli strain SB221 was isolated and transformed into JM109,an E. coli strain with a damaged cell wall (ATCC 53323). Shima, et al.,J. Ferm. Bioeng., 68:75 (1989).

Approximately 2×10⁻³ U of GaIT can be obtained from a 150 mLfermentation, corresponding to a 35-fold increase of activities comparedto the previous expression in SB221. Aoki, et al., supra. The enzymeappeared primarily in the chloroform extracted periplasmic fraction asno significant difference in activity excreted into the media wasobserved.

In a similar mannner, sialyl transferase enzymes can be obtained fromcommercial sources, (Sigma Chemical Company, St. Louis, Mo.; BoehringerMannheim, Indianapolis, Ind. and Genzyme, Cambridge, Mass.), isolatedand purified from animal tissues such as bovine submaxillary glandan_(d) rat liver [See e.g., Gross et al., Eur. J. Biochem., 168:595(1987) and Higa et al., J. Biol. Chem., 260(15):8838 (1985)] or inrecombinant form [See e.g., Ernst et al., J. Biol. Chem., 264:3436(1989); Masibay et al., Proc. Natl. Acad. Sci. U.S.A., 86:5733 (1989);Toghrol et al., Biochemistry, 29:2349 (1990); Appert et al., EMBO,9:3171 (1990) and Joziasse et al., Eur. J. Biochem., 191:75 (1990)].

B. Glycosidase Methods

An alternative synthetic method for the production of theoligosaccharides of the present invention uses the enzymeβ-galactosidase. According to such a method a monosaccharide acceptor ofthis invention is reacted with a β-galactoside derivative. A preferredβ-galactoside derivatives is a β-nitrophenyl β-galactoside.

Thus, for example when Compound 5 from Table 2 is reacted withβ-nitrophenyl β-galactoside in the presence of β-galactosidase (EC3.2.1.23 from E. coli) according to Scheme 6 below, Compound 9 isobtained (Example 1F). ##STR20##

Compound 9 possesses a β1,6 linkage. Thus, in contrast to the methodusing glycosyltransferase, the glycosidase method produces compoundshaving a 1,6 glycosidic linkage.

Not all of the monosaccharide acceptors of the present invention aresuitable substrates for β-galactosidase. For example, when Compound 3 or4 from Table 2 are used as a substrate, no product was obtained. Neitherazasugar was an inhibitor of β-galactosidase. p-Nitrophenylβ-galactoside was hydrolyzed by the enzyme at the same rate in theabsence and presence of the azasugars.

Confirmation of the structure and linkage of synthesized oligosaccharidecompounds is accomplished by nuclear magnetic resonance (NMR)spectroscopy using ¹ H and ¹³ C NMR data (at 300 MHz or 500 MHz, and 125MHz, respectively). The 1H-NMR chemical shift assignment for each protonis established by extensive decoupling experiments.

Typically, a large downfield shift (0.1-0.3 ppm) for the protonsattached to the carbon in a glycosidic linkage relative to the startingmonosaccharide is observed. Correspondingly, the other protonsexperience little shifts. 1H-Nuclear Overhauser effect (NOE) experimentsare used to further confirm the linkage and conformation.

For example, Compound 9 displayed a 4 percent enhancement of one of theH-6 resonances of the 5-thioglucose when the H-1 of galactosewas-irradiated, indicative of the close proximity of the H-6 of the5-thioglucose to the H-1 of the galactose moiety. Additionally, in the¹³ C-NMR spectra, a downfield shift of the C-6 resonance was observed,along with no significant shifts for the other carbon signals. Such dataindicate that Compound 9 has a 1,6 linkage.

Further evidence for the assignment of the regiochemistry of adisaccharide linkage is provided by the analysis of the 1H spectra ofthe per-acetylated disaccharide. Thus, the H-1 to H-4 resonances of a5-thio-D-glucose moiety (Compound 5 from Table 2) experiences a largedownfield shift of 1.3 to 1.8 ppm (relative to the free disaccharide)upon acetylation. Correspondingly, the H-5 and H-6 resonances experienceonly minor shift (-0.33 to +0.38 ppm) upon acetylation.

The Compositions

The present invention also contemplates a composition that comprises aglycosidase- or glycosyltransferase-inhibiting amount of abefore-described oligosaccharide dispersed in an aqueous medium.Preferably, the aqueous medium is a pharmaceutically acceptable,non-toxic medium such as normal saline, phosphate-buffered saline,Ringer's solution or the like as are well known in the art. The aqueousmedium can also comprise blood, serum, plasma or lymph of a mammal suchas a mouse, rat, rabbit, guinea pig, dog or human to which theazapyranose is administered.

A glycosidase- or glycosyltransferase-inhibiting amount is an amountthat inhibits a preselected glycosidase or glycosyltransferase enzyme byat least 25 percent, more preferably by about 50 percent, and mostpreferably by about 75 percent or more.

It appears that a glycosidase inhibitor can be used as a Substrate foranother glycosidase (e.g., β-galactosidase). For example, Compound 5from Table 1 is an inhibitor of β-glucosidase.

Compounds 3, 4, 5 and 6 from Table 1 are potent inhibitors ofexoglucosidases (e.g., β-glucosidase), and their glycosides areinhibitors of endoglucosidases: several natural and synthetic productsof this type are potent endoglycosidase inhibitors. See, e.g., Kajimoto,et al., J. Am. Chem. Soc., 113:6187 (1991); and Liotta, et al., J. Am.Chem. Soc., 111:783 (1989).

Further, Compounds 2i and 2o from Table 1 can inhibitα-1,3/4-fucosyltransferase due to the lack of an appropriately oriented3-OH group. Lowe, et al., Cell, 63:475 (1990). The data of Table la showthat Compound 8 is of similar inhibitory potency to Compound 2i, whereasCompound 10a is more inhibitory than either compound. The FucT used forthese studies was provided by Dr. J. B. Lowe of the University ofMichigan, Ann Arbor, Mich.

Compound 2j from Table 1 is useful for the synthesis of Le^(X)[Galβ1,4(Fucα1,3)GlcNAc] and sialyl Le^(x) [NeuAcα2,3Galβ1,4 (Fucαl, 3)GlcNAc] as described in Scheme 3. A chemical-enzymatic synthesis ofsialyl Le^(x) can be carried out with peracetylation of Compound 2j fromTable 1 followed by selective deprotection of the 3-o-allyl group with aRu catalyst [Corey, et al., J. Org. Chem., 38:3224 (1973)] to liberatethe 3-OH group for fucosylation.

The deoxy-azasugars are known to inhibit glycosidase activity as shownbelow in Table-3.

                  TABLE 3                                                         ______________________________________                                        Glycosidase Inhibition                                                                   Brewer's Yeast (BY)                                                Compound   or Sweet Almond (SA)                                                                           K.sub.i (M)                                       ______________________________________                                        103c       α-Glucosidase (BY)                                                                       1.56 × 10.sup.-3                                       β-Glucosidase (SA)                                                                         7.8 × 10.sup.-4                            117        α-Glucosidase (BY)                                                                       1.78 × 10.sup.-3                                       β-Glucosidase (SA)                                                                         1.4 × 10.sup.-4                            118        α-Glucosidase (BY)                                                                       6.95 × 10.sup.-3                                       β-Glucosidase (SA)                                                                        1.49 × 10.sup.-3                            Controls                                                                      119        α-Glucosidase (BY)                                                                       3.69 × 10.sup.-4                                       Type I (calf liver)                                                                             7.0 × 10.sup.-8                                       β-Glucosidase (SA)                                                                         4.3 × 10.sup.-5                            122        α-Glucosidase (BY)                                                                       >1.0 × 10.sup.-2a                                      β-Glucosidase (SA)                                                                         8.0 × 10.sup.-5                            123        α-Glucosidase (BY)                                                                       8.67 × 10.sup.-6                                       Type I (calf liver)                                                                             1.0 × 10.sup.-6a                                      β-Glucosidase (SA)                                                                         1.8 × 10.sup.-5b                                      α-D-Mannosidase                                                                           4.0 × 10.sup.-4                                       (jack bean)                                                                   β-D-Galactosidase                                                                         No inhibition.sup.c                                          (jack bean)                                                        ______________________________________                                         .sup.a Schweden et al., Arch. Biochem. Biophys., 248:335 (1986)               .sup.b Dale et al., Biochemistry, 24:3530 (1985)                              .sup.c No significant inhibition observed with 10 mM inhibitor in the         assay.                                                                   

A before-described oligosaccharide is dispersed in the aqueous medium.Such dispersal includes suspensions as well as true solutions, which areultimate dispersions, in the aqueous medium.

The following examples illustrate specific embodiments of this inventionand are not limiting of the specification and claims in any way.

EXAMPLES Example 1

Synthesis of Oligosaccharides

A. (β-D-Galactopyranosyl-(1,4)-5-thio-D-glucopyranose), Compound 7 fromTable 1

5-thioglucose, Compound 5 from Table 1, (100 mg, 500 mmol), was reactedwith 5 U of GalT (Sigma Chem. Co., St. Louis, Mo.), UDP-glucose (350 mg,500 mmol), α-lactalbumin (0.1 mg/mL) and UDP-glucose epimerase (10 U) in10 mL of 50 mM sodium cacodylate (pH 7.0) containing 5 mM of MnCl₂. GalThad a specific activity of 4-7 U/mg (1 U=1 mmol of UDP-Gal transferredper minute). The purified enzyme had a reported specific activity of 15U/mg using GlcNAc as an acceptor. The K_(m) value for UDP-Gal is 0.5 mM.The reaction mixture was incubated at 37° C. for two days. The productwas isolated via a Dowex 1 formate column followed by gel filtration(Bio Gel P-2) to give 90 mg of the title compound in 50 percent yield.

¹ H-NMR (D₂ O, 500 MHz) δ4.95 (d, J₁,2 =3 Hz, H-1α5ThioGlc), 4.51 (d,J₁,2 =8 Hz, H-1 Gal), 4.05 (dd, J₅,6',=5 Hz, J₆,6',=12 Hz H-6' 5ThioGlcHz), 3.83-3.9 (H-4 Gal; H-4 5ThioGlc; H-6 5ThioGlc), 3.8 (dd, J₂,3 =9.6Hz, H-2 5ThioGlc), 3.65-3.75 (H-5 Gal, H-6 Gal; H-6'Gal, H-3 5ThioGlc),3.62 (dd, J₂,3,=10 Hz, J₃,4 =3.5 Hz, H-3 Gal), 3.53 (dd, H-2 Gal), 3.32(ddd, J₄,5 =10.5 Hz, J₅,6 =2.5 Hz, J₅,6',=5 Hz, H-5 5thioGlc); ¹³ C-NMR(125 MHz, D₂ O) δ for Gal 103.2 (C-1), 71.7 (C-2), 72.7 (C-3), 69.0(C-4), 75.8 (C-5), 61.5 (C-6); for 5ThioGlc 73.3 (C-1), 75.7 (C-2), 73.0(C-3), 82.3 (C-4), 42.5 (C-5), 59.7 (C-6). HRMS (M+Cs⁺) calcd 490.9988,found 491.0022.

B.β-D-Galactopyranosyl-(1,4)-2-acetamido-6-O-acetyl-2-deoxy-D-glucopyranose,Compound 2e

About 2 0 mg (76 mmol) of 3 -O-AcetylGlcNAc, Compound 1f from Table 1,was reacted with about 1 equivalent of UDPGlc (50.5 mg) in 1 mL of 0.05MNacacodylate/HCl containing 0.05 mM NAD⁺, 10 mM DTT, and 5 mM MnCl₂, pH7.0. UDPGlc epimerase (1 U) and galactosyltransferase (2 U) were added.The mixture was shaken at 37° C. and after two days another 2 U ofgalactosyltransferase were added. After four days the solution waslyophilized and the residue was purified with a silica gel columnchromatography to obtain 6 mg of the title compound.

¹ H NMR (D₂ O) δ1.98 (s, 3H, NAc), 2.08 (s, 3H, OAc), 3.50-3.57 (m, 1H,2-H, (glcNAc), 3.63-3.82 (m, 6H, 2'-H (gal), 3'-H, 4'-H, 5'-H, 3-H,4-H), 3.85-3.97 (m, 2H, 6'-H₂), 4.01 (ddd, J=7 Hz, J=4 Hz, J=2.2 Hz,5-Ha), 4.10-4.19 (m, 5-Hb), 4.21 (dd, J=12 Hz, J=4 Hz, 6-H_(a) a), 4.22(d, J=7.8 Hz, 1-Ha), 4.23 (d, J=7.8 Hz, 1-Hb), 4.29 (dd, J=2.2 Hz, J=12Hz, 6-H_(b) b), 4.35-4.48 (m, 6-H₂ b). ¹³ C NMR δ21.0, 22.7 (2CH₃),54.4, 56.8 (C-2'), 61.8, 63.8 (C-6, C-6'), 69.3, 70.1 71.7, 73.3 76.3(C-2', C-3', C-4', C-5', C-3, C-4, C-5), 79.4 (C-4), 91.4, 96.1 (C-1),104.0 (C-1'), 174.5, 175.0 (2CO). HRMS calcd. for (C₁₂ H₂₇ O₁₂ N+Cs⁺):558. 0588; found: 558.0590.

C. Allylβ-D-galactopyranosyl-(1,4)-2-acetamido-2,3-dideoxy-β-D-glucopyranoside,Compound 2i

Allyl-1,3-deoxy-GlcNAc, Compound 1i from Table 1, was reacted withUDPGlc in 1 mL of 0.05M Nacacodylate/HCl containing 0.05 mM NAD⁺, 10 mMDTT, and 5 mM MnCl₂, pH 7.0. UDPGlc epimerase (1 U) andgalactosyltransferase (2 U) were added. The mixture was shaken at 37° C.and after two days an other 2 U galactosyltransferase were added. Afterfour days the solution was lyophilized and the residue was purified witha silica gel column chromatography to obtain the title compound.

¹ H NMR (D₂ O) δ1.62 (1H, q, J=12.18 Hz, H-3ax), 1.94 (3H, s, NHAc),2.46 (1H, br dt, J=4.61, 12.57 Hz, H-3eq), 3.44 (1H, dd, J=8.04, 9.92Hz, H-2'), 4.41 (1H, d, J=7.82 Hz, H-1'), 4.51 (1H, d, J=8.38 Hz, H-1);¹³ C NMR (D₂ O) δ22.45, 35.78, 49.23, 61.03, 61.26, 68.89, 70.55, 71.26,73.03, 74.19, 75.52, 78.85, 102.01, 104.21, 118.60, 133.81, 174.12; HRMSCalcd for C₁₇ H₂₉ NO₁₀ Cs (M+Cs⁺): 540.0846. Found: 540.0846.

D. β-D-Galactopyranosyl-(1,4)-D-glucal,

Compound 8

Compound 6 from Table 1, was reacted with UDPGlc in 1 mL of 0.05MNa-cacodylate/HCl containing 0.05 mM NAD⁺, 10 mM DTT, and 5 mM MnCl₂, pH7.0. UDPGlc epimerase (1 U) and galactosyltransferase (2 U) were added.The mixture was shaken at zero degrees C and after four days another 2 Uof galactosyltransferase were added. After four days the solution waslyophilized and the residue was purified with a silica gel columnchromatography to obtain the title compound.

¹ H-NMR (500 MHz, D₂ O) δ6.4 (dd, J₁,2 =6 Hz, J₁,3 =1.6 Hz, H-1 Glucal),4.7 (dd, J₂,3 =2.6 HZ, H-2 Glucal) 4.49 (d, J₁,2 =7.8 Hz, H-1 Gal), 4.35(br dt, J₂,3 =2.6 Hz, J₃,4 =6.5 Hz, H-3 Glucal), 3.99 (d, J₄,5 =9.3 Hz,J₅,6 =J₅,6',=3.7 HZ, H-5 Glucal), 3.85-3.9 (H-4 Gal; H-6 and H-6'Glucal), 3.82 (dd, H-4 Glucal), 3.68-3.75 (H-5, H-6, H-6' Gal), 3.63(dd, J₂,3 =10 Hz, J₃,4 =3.4 Hz, H-3 Gal), 3.5 (dd, J₁,2 =8.6 Hz, H-2Gal). ¹³ C-NMR (125 MHz, D₂ O) δ for Gal 103.9 (C-1), 71.9 (C-2), 73.5(C-3), 69.5 (C-4), 76.3 (C-5), 42.0 (C-6); for glucal 144.9 (C-1), 102.7(C-2) 68.3 (C-3), 78.4 (C-4), 77.7 (C-5), 60.6 (C-6). HRMS (M+Cs⁺)calcd. 441. 0162, found 441. 0121.

E. β-D-Galactopyranosyl-(1,4)-deoxynojirimycin, Compound 10a

Compound 6 from Table 1, was reacted with UDPGlc in 1 mL of 0.05MNa-cacodylate/HCl containing 0.05 mM NAD⁺, 10 mM DTT, and 5 mM MnCl₂, pH7.0. UDPGlc epimerase (1 U) and galactosyltransferase (2 U) were added.The mixture was shaken at 37° C. and after four days another 2 U ofgalactosyltransferase were added. After four days the solution waslyophilized and the residue was purified with a silica gel columnchromatography to obtain the title compound.

¹ H-NMR (D₂ O, 500 MHz) δ4.3 (d, J₁,2 =7.5 Hz, H-1 Gal), 3.76 (dd,J₅,6',=3.0 Hz, J₆,6',=12.5 Hz, H-6' DNJ), 3.74 (br d, J₃,4 =Hz, H-4Gal), 3.7 (dd, J₅,6 =5.0 Hz, H-6 DNJ), 3.52-3.65 (m, H-6 Gal, H-6' Gal,H-5 Gal, H-2 DNJ, H-4 DNJ), 3.5 (dd, J₂,3 10.5 Hz, H-3 Gal), 3.39 (t,J₂,3 =J₃,4 =9.5 Hz, H-3 DNJ), 3.38 (dd, H-2 Gal), 3.13 (dd, J_(1eq),1ax=12.5 Hz, J_(1eg),2 =5.0 Hz, H-1eq DNJ), 2.85-2.90 (m, H-5 DNJ), 2.56(br t, J_(1ax),2 =12 Hz, H-1ax DNJ). ¹³ C-NMR (125 Hz, D₂ O) δ for Gal103.7 (C-1), 71.7 (C-2), 73.2 (C-3), 69.2 (C-4), 76.3 (C-5), 61.8 (C-6);for DNJ 47.4 (C-1), 69.04 (C-2), 76.2 (C-3), 78.9 (C-4), 59.4 (C-5),60.0 (C-6). HRMS (M+Cs⁺) calcd. 458.0427, found 458.04444.

F. β-D-Galactopyranosyl-(1,6)-5-thio-D-glucopyranose, Compound 9

β-Galactosidase from E. coli (EC 3.2.1.23:0.50 mg, 172 U) was added at23° C. to a solution of 4-nitrophenyl β-D-galactopyranoside (150 mg,0.50 mmol) and 5-thio-D-glucose (49 mg, 0.25 mmol) in Na₂ HPO₄ /MgCl₂buffer (4 mL of a 0.10M solution in Na₂ HPO₄ and 10 mM in MgCl₂, pH 7.0)and Tris (1 mL of a 0.05M solution, pH 7.3). The reaction was maintainedat 23° C. with periodic monitoring by TLC. After 58 hours, the reactionwas terminated by heating at 100° C. for 30 minutes. The solution wasfiltered and lyophilized and the residue was purified by columnchromatography (silica gel, 3:2:1 ethyl acetate-acetic acid-water). Thefraction containing the disaccharide was further purified by gelfiltration chromatography using a Bio-Gel P-2 column (2×40 cm, 200-400mesh) eluted with H₂ O to afford the title compound (26.4 mg, 29.5percent based on 5-thio-D-glucose) as a white amorphous solid (Rf=0.44,silica gel, 3 2:1 EtOAc-HOAc-H₂ O). The silica gel chromatography alsoafforded galactose and 5-thio-D-glucose. Analysis of the disaccharideindicated a mixture of a to b anomers in a ratio of 11:1. A Anomer: ("A"refers to the 5-thioglucose moiety while "B" refers to the galactosemoiety.) The differences in coupling constants are due to round-offerror.)

¹ H NMR (500 MHz, D₂ O) δ4.82 (d, J=3.0 Hz, 1H, H1-A), 4.22 (d, J=8.0Hz, 1H, H1-B), 4.01 (dd, J=2.5, 11.0 Hz, 1H, H6-A), 3.82 (dd, J=5.5,11.0 Hz, 1H, H6-A), 3.74 (d, J=3.0 Hz, 1H, H4-B), 3.63-3.54 (m, 3H,H2-A, H3-A, H5-B), 3.50 (t, J=10.5 Hz, 1H, H4-A), 3.48-3.45 (m, 2H,H6-B), 3.46 (dd, J=3.5, 10.0 Hz, 1H, H3-B), 3.36 (app. t, J=9.0 Hz, 1H,H2-B), 3.20-3.14 (m, 1H, H5-A); ¹³ C NMR (125 MHz, D₂ O) δ103.8, 75.6,74.0, 73.7, 73.6, 73.1, 71.1, 69.1, 68.8 (CH₂), 61.4 (CH₂), 41.6; exactmass calcd for C₁₂ H₂₂ O₁₀ SCs (M+Cs⁺) 490.9988, found 491.0013.

G. β-D-Galactopyranosyl-(1,6)-5-thio-D-glucopyranose octaacetate

Pyridine (0.9 mL, 11.1 mmol), Ac₂ O (0.14 mL, 1.48 mmol), and (GlcNAc)₂Table 1 (33 mg, 0.09 mmol) were combined at 0° C. The reaction mixturewas allowed to warm to 23° C. maintained for 23 hours, and diluted withethyl acetate (10 mL). The organic phase was rinsed with 1N HCl (10 mL)and the acidic fraction was extracted with ethyl acetate (2×20 mL). Thecombined organic phases were rinsed with brine (10 mL), dried (MgSO₄),and concentrated. The residue was purified by column chromatography(silica gel, 3:1 EtOAc-toluene) to afford the per-acetylated titledisaccharide (24 mg, 93 percent) as a colorless glass (Rf=0.61, silicagel, 3:1 EtOAc-toluene). Analysis of the disaccharide indicated amixture of α to β anomers in a ratio of 6:1.

α Anomer: ¹ H NMR (500 MHz, CDCl₃) δ6.12 (d, J=3.5 Hz, 1H, H1-A), 5.42(app t, J=10.0 Hz, 1H, H3-A), 5.38 (dd, J=1.0, 3.5 Hz, 1H, H4-B), 5.21(dd, J=3.0, 10.0 Hz, 1H, H2-A), 5.19 (app t, J=11.0 Hz, 1H, H4-A), 5.18(dd, J=8.0, 10.5 Hz, 1H, H2-B), 4.99 (dd, J=3.5, 10.5 Hz, 1H, H3-B),4.40 (d, J=9.0 Hz, 1H, H1-B), 4.16 (dd, J=6.5, 11.0 Hz, 1H, H6-B), 4.10(dd, J=6.5, 11.0 Hz, 1H, H6-B), 4.05 (dd, J=3.5, 10.0 Hz, 1H, H6-A),3.88 (dt, J=1.0, 7.0 Hz, 1H, H5-B), 3.57 (ddd, J=3.5, 7.0, 10.5 Hz, 1H,H5-A), 3.49 (dd, J=7.5, 10.5 Hz, 1H, H6-A), 2.18 (s, 3H, OAc), 2.14 (s,3H, OAc), 2.09 (s, 3H, OAc), 2.052 (s, 3H, OAc), 2.046 (s, 3H, OAc),2.01 (s, 3H, OAc), 1.99 (s, 3H, OAc), 1.98 (s, 3H, OAc); ¹³ C NMR (62MHz, CDCl₃) δ170.3, 170.2, 170.1, 169.7, 169.6, 169.4, 169.1, 101.2,73.1, 72.3, 70.8, 70.4, 68.1, 67.3 (CH₂), 66.8, 61.0 (CH₂), 40.8, 20.9,20.8, 20.6, 20.5; exact mass calcd for C₂₈ H₃₈ O₁₈ SCs (M+Cs⁺) 827.0833,found 827.0823.

These syntheses demonstrate that Gat. T and β-galactosidase can be usedas catalyst for the preparation of disaccharides on milligram scaleswith weak acceptor substrates. Given that the enzyme is relativelystable, GalT (and perhaps other glycosyltransferases) seems amenable forthe small-scale synthesis of a number of unusual oligosaccharides.

All the subject disaccharides possess similar glycosidic torsional angleas evidenced by the significant NOE's (6-10 percent) between H-1' andH-4. This observation is consistent with that reported by Lemieux thatthe glycosidic torsional angle is mainly determined by the exo-anomericeffect.

Example 2

Synthesis of Sialyl N-Acetyllactosamine

Sialyl N-acetyllactosamine (NeuAcα2,6Galβ1,4GlcNAc) was synthesized inan enzyme-catalyzed method with the in situ regeneration of CMP-sialicacid according to scheme 4 set forth above.

Neuraminic acid (NeuAc), CMP, ATP, PEP (monosodium salt), MgCl₂.6H₂ O,MnCl₂.4H₂ O, KCl, pyruvate kinase (PK, EC 2.7.1.40), nucleosidemonophosphate kinase (NMK, EC 2.7.4.4) and inorganic pyrophosphatase(PPase, EC 3.6.1.1) were purchased from Sigma Chemical Co., St. Louis,Mo. Siaα2,6Gal sialyl transferase (EC 2.4.99.1) was obtained as agenerous gift and can be purchased from Sigma Chemical Co., St. Louis,Mo. CMP-NeuAc synthetase (EC 2.7.7.43) was obtained from E. colitransformed with a CMP-NeuAc gene according to the method set forthbelow.

NeuAc (0.92 g, 3 mmol), Galβ4GlcNAc (1.1 g, 3 mmol), CMP (0.1 g, 30μmol), ATP (16 mg, 3 μmol), PEP (2.8 g, 6 mmol), MgCl₂.6H₂ O (0.61 g, 3mmol), MnCl₂.4H₂ O (0.15 g, 0.8 mmol), KCl (0.22 g, 3 mmol), NMK or MK(450 U), PK (6,000 U), PPase (300 U), CMP-NeuAc synthetase (24 U), andSiaα2,6Gal sialyl transferase (4 U) were mixed with 150 ml of HEPESbuffer (0.2M, pH 7.5) to form a reaction mixture and the reactionmixture maintained under argon at about 25° C. for about 48 hours. Afterthe disappearance of NeuAc (determined by thin-layer chromatography) thereaction mixture was reduced in volume to 20 ml by lyophilization andthe lyophilized reaction mixture applied to a Bio Gel P2 (200-400 mesh)column with water as the mobile phase. The trisaccharide-containingfractions were eluted, collected and lyophilized to give pureNeuα2,6Galβ1,4GlcNAc in 97 percent yield.

¹ H-NMR δ1.701 (1H, t, J=12.5 Hz, H-3_(ax) of NeuAc), 2.007 (3H, s, NHAcof GlcNAc), 2.004 (3H, s, NHAc of NeuAc), 2.649 (1H, dd, J=5.0 and 12.5Hz, H-3_(eq) of NeuAc, 4.43 1,d, J=8.0 Hz, H-1 of Gal), 4.73 (0.5H, d,J=8.0 Hz, H-1b of GlcNAc), and 5.178 (0.5H, d, J=2.5 Hz, H-1a ofGlcNAc).

The turn-over number for ATP was about 1000 and that of CMP, CDP, CTPand CMP-NeuAc was about 100.

These data show that a glycosyl compound can be sialylated in anefficient, enzyme-catalyzed, self-contained, cyclic, synthetic methodinvolving the in situ regeneration of CMP-sialic acid. This syntheticmethod provides a novel, high-yield (97 percent) scheme for thelarge-scale preparation of sialylated glycosyl compounds.

A. Preparation of Recombinant CMP-NeuAc Synthetase

The gene coding for CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase(EC 2.7.7.43) was amplified from total DNA of E. coli strain K-235through a primerdirected polymerase chain reaction. The gene was fusedwith a modified ribosome binding site of the original CMP-NeuAcsynthetase gene and a decapeptide tag sequence which served as a markerfor screening of expressed proteins. The gene was cloned into lambdaZAP™ vector at Eco RI and Xba I sites and overexpressed in E. coli Sureat a level approximately 1000 times that of the wild type.

E. coli strain K235 (ATCC 13207) was obtained from American Type CultureCollection and maintained on LB (Luria-Bertani) medium (one litercontains: Bacto Tryptone, 25 g; Yeast extract, 10 g; NaCl, 3 g: pH 7.0).Genomic DNA was extracted from the E. coli according to the methoddescribed by Maniatis et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989).

The CMP-NeuAc synthetase gene was isolated via PCR amplification in thepresence of two custom-designed primers (Table 4).

                                      TABLE 4                                     __________________________________________________________________________     ##STR21##                                                                     ##STR22##                                                                    __________________________________________________________________________

Primer CMP5 contained an Eco RI restriction site, a ribosomal bindingsequence, a start codon and an oligonucleotide corresponding to theN-terminal hexapeptide of the enzyme (underlined above). Primer CMP3contained an Xba I restriction site, a-stop codon, a decapeptide tagsequence and a sequence corresponding to the C-terminal heptapeptide ofthe enzyme (also underlined above).

PCR amplification was performed in a 100 μL reaction mixture containing2 μL (2 μg) of E. coli strain K235 DNA, 400 nmol of primers CMP5 andCMP3, 200 μM of different dNTPs, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 2mM MgCl₂, 0.01 percent (w/v) gelatin, 0.1 percent (v/v) Triton X-100,and 2 units of Thermus aquaticus DNA polymerase. The reaction wasoverlaid with mineral oil and subjected to 35 cycles of amplification.The cycle conditions were set as denaturation at 94° C. for one minute,annealing at 60° C. for two minutes, and elongation at 72° C. for 1.5minutes. The primers were annealed with E. coli DNA at 94° C. for twominutes followed by slow cooling to room temperature prior to PCRamplification.

The amplified gene was cloned into lambda ZAP™ II vector at the Eco RIand Xba I sites for the construction of phagemid for expression of theenzyme in E. coli.

Lambda Zap™ is a derivative of the original Lambda Zap™ vector (ATCC#40,298) that maintains all of the characteristics of the originalLambda Zap™ including 6 unique cloning sites, fusion protein expression,and the ability to rapidly excise the insert in the form of a phagemid(Bluescript SK-), but lacks the SAM 100 mutation, allowing growth onmany Non-Sup F strains, including XL1-Blue. The Lambda Zap™ II vectorwas constructed as described by Short et al., [Nucleic Acids Res.,16:7583 (1988)] by replacing the Lambda S gene contained in a 4254 basepair (bp) DNA fragment produced by digesting Lambda Zap™ with therestriction enzyme Nco I. This 4254 bp DNA fragment was replaced withthe 4254 bp DNA fragment containing the Lambda S gene isolated fromLambda gt10 (ATCC #40,179) after digesting the vector with therestriction enzyme Nco I. The 4254 bp DNA fragment isolated from lambdagt10 was ligated into the original Lambda Zap™ vector using T4 DNAligase and standard protocols for such procedures described in CurrentProtocols in Molecular Biology, Ausubel et al., eds., John Wiley andSons, New York, 1987.

The DNA obtained from the PCR amplification was purified on 0.6 percentagarose gel. The DNA band corresponding to 1.3 kb was separated from theagarose and electro-eluted. The DNA was then extracted withphenol/chloroform and precipitated with ethanol overnight at 20° C. Theprecipitated DNA was disclosed in a proper restriction enzyme buffersupplied by Boehringer Mannheim Biochemical Co. (Indianapolis, Ind.) anddigested with 40 units/μg DNA of Eco RI and Xba I at 37° C. for twohours. The digested DNA was then recovered by phenol/chloroformextraction and ethanol precipitation and resuspended in a TE buffer (pH7.5). This DNA was used as an insert. The arms were also prepared fromthe digestion of vector lambda Lcl with 20 Units/mg DNA of Eco RI andXba I and recovered with ethanol precipitation after extraction withphenol/chloroform. Vector lambda LcI was obtained as a generous giftfrom Dr. R. A. Lerner (Scripps Clinic and Research Foundation, La Jolla,Calif.). The insert was then ligated with the arms and packaged with apackaging kit as suggested by the manufacturer (Stratagene Co., SanDiego, Calif.). The PCR amplification product insert with lambda Lclarms is shown in FIG. 2.

After packaging, the phage solution was used to infect host strainXL1-Blue (Stratagene Co., San Diego, Calif.) and plated on LB agarplates at 37° C. After plaque formation was observed, a nitrocellulosemembrane previously soaked with 0.5 mM IPTG(Isopropyl-β-D-thiogalactopyranoside) solution was carefully placed onthe top of the agar and incubated at 25° C. overnight (about 15 hours).The nitrocellulose membrane was then used for screening with alkalinephosphatase conjugated with the anti-decapeptide tag antibody. Positiveclones with intense blue color plaques were cored from the agar platesand transferred to sterile microfuge tubes containing 500 μL of SMbuffer and 20 μl of CHCl₃. For extension, 200 μl of the phase stock wasmixed with 200 μL of LX1-Blue cells (OD₆₆₀ =1.0) and 2 μL of R408 helperphage (1×10¹¹ pfu/ml from Stratagene Co.), and the mixture incubated at37° C. for 10 minutes. Excised plasmids were used to infect XL1-Bluecells [Short et al. Nucleic Acids Res., 16:7583 (1988)], plated, andanalyzed for expression of the enzyme by ELISA and enzyme activityassays.

One clone which produced higher CMP-NeuAc synthetase was isolated anddesignated as strain SIL-B3 and the phagemid contained thereindesignated CMPSIL-1 (FIG. 3). Phagemid CMPSIL-1 was isolated from strainSIL-B3 using a plasmid isolation kit (Qiagen Inc., Studio City, Calif.)and transformed to E. coli Sure competent cells (Stratagene Co., SanDiego, Calif.). Transformed cells were plated on LB agar platescontaining 250 μg/mL ampicillin and screened for high enzyme productionusing the ELISA assay. One strain, designated E. coli SIL-S22 (ATCC68531), produced about 100 units of CMP-NeuAc synthetase activity perliter of culture broth. This production of CMP-NeuAc synthetase is over1000 times greater than the amount of CMP-NeuAc synthetase produced bywild-type, non-transformed E. coli and over 30 timers greater than theamount-of CMP-NeuAc synthetase produced by the transformed cellsdescribed by Zapata et al., J. Biol. Chem., 264(25):14769 (1989).

E. Coli strain SIL-S22 were grown on LB-rich medium containing 250 μg/mLampicillin to mid-logarithmic phase (OD₆₆₀ about 0.6-0.7) and inducedwith 0.5 mM IPTG for 10 hours at 30° C. The culture broth wascentrifuged at 10,000×g for 20 minutes at 4° C. and the resulting cellpellet was washed with a buffer containing 0.2M Tris (pH 7.5), 0.2 mMdithiothreitol and 20 mM MgCl₂. After washing, the cell pellet wasresuspended in the same buffer and disrupted by a French pressure cellat 16,000 lb/in² and centrifuged at 23,000×g for 60 minutes. Theresulting supernatant was assayed for enzyme activity assay according tothe method of Vann et al., J. Biol. Chem., 262:17556 (1987) except thatthe developed color was extracted with cyclohexanone.

The enzyme was incubated in a 250 μl buffer containing 5.5 mM CTP, 2.8mM N-acetylneuraminic acid, 0.2M Tris, 20 mM MgCl₂ and 0.2 mM DTT, pH9.0. After the mixture was incubated at 37° C. for 30 minutes, 50 μl of1.6M NaBH₄ was added to destroy excess NeuAc at room temperature for 15minutes. The mixture was then put in ice bath and 50 μl of H₃ PO₄ wasadded to destroy NaBH₄. The mixture was kept at zero degrees C for fiveminutes then incubated at 37° C. for 10 minutes to cleave thephosphoester bond of the formed CMP-N-acetylneuraminic acid. The freeN-acetylneuraminic acid was oxidized with 50 μl of 0.2M NaIO₄ at roomtemperature for 10 minutes, and 400 μl of 4 percent NaAsO₂ in 0.5 N HClwas added. The solution mixture was then transferred to a test tubecontaining 1 ml of 0.6 percent thiobarbituric acid in 0.5M Na₂ SO₄, andheated in boiling water for 15 minutes. After the solution was cooled, 1ml of the solution was taken out and mixed with 1 ml of cyclohexanone.The mixture was shaken and centrifuged, and the upper layer was takenfor the measurement at 549 nm.

CMP-NeuAc was isolated by affinity chromatography using theanti-decapeptide antibody or Orange A (Amicon C., Danvers, Mass.) asligand followed by gel filtration. Huse et al., Science, 246:1275(1989). The cell free extract (30 mL) obtained as described above waspassed through an Orange A Dye column (1.5 mg/ml gel, 3 cm×30 cm) andwashed with 200 ml of Tris buffer (0.2M Tris, 0.2 mM DTT and 2 mM MgCl₂,pH 7.5). The enzyme was eluted with a linear gradient from 0M KCl to 1MKCl in the same buffer. The active fraction was pooled and concentratedto 5 ml by ultrafiltration. The concentrated enzyme solution was thenpassed through an FPLC gel filtration column (Superose 12 h 10/30,Pharmacia Co.) at a flow rate of 0.2 ml/minute and the active fractionswere collected. The protein concentration was determined by BCA assaykit (Pierce Co., Rockford, Ill.). The purity of the protein was judgedby SDS PAGE (Phastsystem, Pharmacia Co.).

Example 3

Synthesis of a Sialyl Trisaccharide

Two procedures have been combined to synthesize a sialyl trisaccharide.The enzymatic aldol reaction (cycle B in Scheme 2) was first introducedto the Scheme 1: ManNAc was converted to NeuAc catalyzed by NeuAcaldolase (EC 4.1.3.3) in the presence of pyruvic acid. Although NeuAcaldolase also catalyzes the reverse reaction (NeuAc to ManNAc andpyruvate), the produced NeuAc is irreversibly incorporated into cycle Aof Scheme 2 via CMP-NeuAc catalyzed by CMP-sialic acid synthetasecoupled with inorganic pyrophosphatase (PPase)-catalyzed decompositionof the released inorganic pyrophosphate. The sialyl LacNAc-was obtainedin 89 percent yield after a Bio-Gel P-2 column chromatography. Theexperimental procedure is as follows:

To a 1.65 mL of HEPES buffer (200 mM, pH 7.5) were added ManNAc (43 mg,180 mmol), LacNAc (22 mg, 60 mmol), CMP (2.0 mg, 6 mmol), ATP (0.32 mg,0.6 mmol), PEP sodium salt (56 mg, 240 mmol), MgCl₂ •6H₂ O (12.2 mg, 60mmol), MnCl₂ •4H₂ O (3.0 mg, 16 mmol), KCl (4.4 mg, 60 mmol), pyruvicacid sodium salt (33 mg, 300 mmol), NeuAc aldolase (EC 4.1.3.3; 45 U),MK (EC 2.7.4.3; 100 U), PK (EC 2.7.1.40; 120 U), PPase (EC 3.6.1.1; 6U,mercaptoethanol (0.22 mL), CMP-NeuAc synthetase (EC 2.7.7.43; 0.3 U in 1mL of 0.1M Tris buffer, pH 9), and a(2,6)sialyltransferase (EC 2.4.99.1;0.08 U). The final volume of the reaction mixture was 3 mL. The reactionwas conducted at room temperature for two days under argon. Afterdisappearance of the starting material judged by TLC (R_(f) : LacNAc,0.63; NeuAc, 0.31; sialyl LacNAc; 0.30; CMP-NeuAc, 0.19 in 1M NH₄OAc/iPrOH 1:2.4, v/v), the reaction mixture was directly applied on aBio-Gel P-2 (200-400 mesh) column (2×36 cm) and eluted with water. Thetrisaccharide-containing fractions were pooled and lyophilized to givesialyl LacNAc (37 rag, 89 percent)

The LacNAc synthesizing cycle (C in Scheme 2) and the above-mentionedcycle (A+B in Scheme 2) were also combined. The experimental procedureis as follows:

To a 2.6 mL of HEPES buffer (200 mM, pH 7.5) was added ManNAc (43 mg,180 mmol), GlcNAc (13.3 mg, 60 mmol), Glc-i-P (21.5 mg, 60 mmol), CMP(2.0 mg, 6 mmol), UDP (2.8 mg, 6 mmol), ATP (0.32 mg, 0.6 mmol), PEPsodium salt (75 mg, 320 mmol), MgCl₂ •6H₂ O (16.3 mg, 80 mmol), MnCl₂•4H₂ O (4.0 mg, 20 mmol), KCl (6.0 mg, 80 mmol), pyruvic acid sodiumsalt (33 mg, 300 mmol) NeuAc aldolase (45 U), (100 U), PK (120 U), PPase(12 U), mercaptoethanol (0.33 mL), galactosyl transferase (EC 2.4.1.22;1 U), UDP-Glc pyrophosphorylase (EC 2.7.7.9; 1 U), UDP-Gal 4-epimerase(EC 5.1.3.2; 1 U), CMP-NeuAc synthetase (0.3 U in 1 mL of 0.1M Trisbuffer, pH 9), and a (2,6) sialyltransferase (0.08 U). The final volumeof the reaction mixture was 4 mL. The reaction was complete in two days,and pure sialyl LacNAc (9 mg; 22 percent) was isolated based on theabove-mentioned procedure.

These data demonstrate the efficient synthesis of a sialyltrisaccharides starting from GlcNAc, ManNAc, Glc-i-P, and catalyticamounts of CMP, UDP (0.1 equivalents each) and ATP (0.01 equivalent)with no tedious separate preparations of sugar nucleotides, which areregenerated in situ. The pyruvate generated from PEP is used as asubstrate in the NeuAc aldolase reaction.

Example 4

Synthesis of Monosaccharide Acceptors

A. 2-Acetamido-3-O-acetyl-2-deoxy-D-glucopyranose, Compound 1f

The synthesis of 2-acetamido-3-O-acetyl-2-deoxy-D-glucopyranose,Compound 1f, was accomplished by the scheme outlined below. ##STR23##

Compound 12 (2 g, 5 mmol ), prepared from Compound 11 by standardprocedures, was dissolved in 20 mL dry pyridine and treated with fourequivalents (2.6 g) of acetic anhydride. The mixture was refluxed for 10hours, then quenched with ice and extracted with chloroform. The extractwas washed with 2N HCl (2×100 mL), water (50 mL) and brine (50 mL).After being dried over MgSO₄ and evaporation, the product, benzyl2-acetamido-3-O-acetyl-4,6-O-benzylidene-2-deoxy-α-D-glucopyranoside,Compound 13a, was crystallized from ethyl acetate to yield 1.43 g (65percent).

1H NMR (d-6 acetone) δ1.93 (s, 3H, NHAc), 2.06 (s, 3H, COCH₃), 3.72-4.28(m, 5H, H-2, 4, 5, 6a, 6b), 4.44, 4.68 (2d, J=14 Hz, 2H, CH₂ Ph), 4.79(d, J=4 Hz, 1H, H-1), 5.08 (s, 1H, benzylic), 5.27 (dd, J=10 Hz, J=9.5Hz, 1H, 3-H), 7.25-7.40 (m, 5H, Ar), 8.50 (d, J=9 Hz, 1H, NH).

Compound 13a (800 mg, 2 mmol) was dissolved in 100 mL of ethanol and 20mL of acetic acid and the mixture was hydrogenated under 50 psi at roomtemperature using 250 mg of five percent Pd/C. After filtration throughCelite, evaporation, and column chromatography, Compound 1f (350 mg, 67percent) was obtained and further crystallized from methanol/ethylether.

¹ H NMR (D₂ O) δ1.94 (s, 3H, OAc), 2.06 (s, 3H, NAc), 3.36 (ddd, J=9, 5,and 2.5 Hz, H-5b),-3.51 (t, J=9.5 Hz, H-4b), 3.56 (t, J=9.5 Hz, H-4a),3.72 (dd, J=12, 5 Hz, H-6a), 3.78 (dd, J=12, 2.5 Hz, H-6_(b) a), 3.87(ddd, J=9.5, 5 and 2.5 Hz, H-5a), 4.05 (dd, J=10.5, 3.5 Hz, H-2a), 4.72(d, J=9 Hz, H-1b), 4.95 (dd, J=10.5, 9 Hz, H-3b), 5.07 (d, J=3.5 Hz,H-1a), 5.18 (dd, J=10.5, 9 Hz, H-3a). ¹³ C NMR (D₂ O) δ21.3, 22.7(2CH₃), 53.2 (C-2), 61.3 (C-6), 68.7, 72.3, 74.8, 76.7 (C-3, C-4, C-5),91.9, 95.9 (C-1), 174.8, 175.3 (2CO).

B. 2-Acetamido-2-deoxy-3-O-propyl-D-glucopyranose, Compound 1h

The synthesis of Compound 1h was accomplished by the scheme outlinedbelow. ##STR24##

Compound 12 (2 g, 5 mmol) was dissolved in 30 mL THF. NaH (240 mg, 60percent mineral oil dispersion, 1.2 equivalent) was added at zerodegrees C and subsequently followed by 0.52 mL of allyl bromide (1.2equivalents). The mixture was heated to reflux for 12 hours and thenquenched with ice and NH₄ Cl solution. After extraction with water (100mL) and brine (50 mL), dried over MgSO₄ and evaporated.Recrystallization from ethyl acetate/hexane gave 1.32 g of Benzyl2-acetamido-3-O-allyl-4,6-O-benzylidene-2-deoxy-α-D-glucopyranoside,Compound 13b (60 percent).

¹ H. NMR (d-6 acetone) δ1.85 (s, 3H, NHAc), 3.60-4.30 (m, 8H, H-2, 3, 4,5, 6a, 6b, CH₂ of allyl), 4.50, 4.72 (2d, J=15 Hz, CH₂ Ph), 4.81 (d, 1H,J=4 Hz, 1H, H-1), 5.03-5.25 (m, 2H, CH₂ ═C of allyl), 5.13 (s, 1H,benzylic), 5.75-5.96 (m, 1H, CH═of allyl), 7.30-7.50 (m, 5H, Ar), 8.12(d, J=9 Hz, 1H, NH).

Compound 13b (440 rag) was dissolved in 10 mL of ethanol and 5 mL ofcyclohexane. 100 mg of PdO was added and the mixture was refluxed for 16hours. The catalyst was removed by filtration and the filtrate wasevaporated. After column chromatography with silica gel (CHCl₃/methanol/hexane 6:2:1), Compound 1h was obtained (120 mg, 46 percent).

¹ H NMR (D₂ O) δ0.92 (t, J=7 Hz, 3H, CH₃), 1.56 (m, 2H, H-2'_(x)), 1.96(s, 3H, NHAc), 3.23-3.85 (m, 7H, H-3, 4, 5, 6, 1'), 3.93 (dd, J=9 Hz,J=4 Hz, H-2a) 4.58 (d, J=8 Hz, H-1b), 5.02 (d, J=4 Hz, H-1a).

C. Methyl 2-acetamido-3-O-allyl-2-deoxy-α-D-glucopyranoside, Compound 1g

The synthesis of Compound 1g was accomplished by the scheme outlinedbelow. ##STR25##

Compound 16 (500 mg, 1.15 mmol) prepared from Compound 14 via Compound15 by standard protection and alkylation methods, was dissolved in 4 mLof concentrated acetic acid. The solution was stirred for 12 hours at80° C. After evaporation of the acetic acid the residue was purified bycolumn chromatography (ethyl acetate/methanol 20:1) on silica gel toyield 157 mg (50 percent) of Compound 1g.

¹ H NMR (d-5 pyridine) δ2.16 (s, 3H, NHAc), 3.32 (s, 3H, Ar--OMe), 3.66(s, 3H, C-1-OMe), 3.80-4.08 (m, 5H, 3-H, 4-H, 5-H, 6-H₂), 4.35 (dd,J=9.5 Hz, J=4.5 Hz, 1H, 2-H), 4.16-4.60 (m, 2H, CH₂ of allyl), 4.78-4.90(m, 1H, 5-H), 5.04-5.40 (m, 2H, CH₂ ═C), 5.74 (s, 1H, benzylidene),5.90-6.10 (m, 1H, CH═C of allyl), 7.00-7.70 (m, 4H, ar), 8.95 (d, J=9Hz, NH).

D. Allyl 2-acetamido-2,3-dideoxy-β-D-glucopyranoside, Compound 1i

The synthesis of Compound 1i was accomplished by the scheme outlinedbelow. ##STR26##

A mixture of allyl 2,3-dideoxy-2-phthalimido-β-D-glucopyranoside,Compound 22, (100 mg, 0.30 mmol) and BuNH₂ (4 mL) in MeOH (20 mL) wasrefluxed for 10 hours, then cooled and evaporated. Acetic anhydride (2mL) was added to a solution of the residue in MeOH (10 mL) at 0°-5° C.,and the mixture was stirred for three hours at 0°-5° C. The mixture wasconcentrated, and the residue was triturated in MeOH with Et₂ O to giveCompound 1i (40 mg, 54 percent).

¹ H NMR (D₂ O) δ1.52 (1H, q, J=12.35 Hz, H-3ax), 1.98 (3H, s, NHAc),2.25 (1H, dt, J=4.76, 12.38 Hz, H-3eq), 3.39 (1H, ddd, J=2.30, 6.44,9.45 Hz, H-5), 3.59 (1H, dt, J=4.79, 9.45 Hz, H-4), 3.65 (1H, dd,J=6.44, 12.30 Hz, H-6a), 3.73 (1H, ddd, J=4.76, 8.45, 12.90 Hz, H-2),3.84 (1H, dd, J=2.30, 12.30 Hz, H-6b), 4.49 (1H, J=8.45 Hz, H-1); ¹³ CNMR (D₂ O) δ22.30, 36.52, 49.24, 61.22, 64.57, 80.09, 102.03, 118.54,133.67, 174.03; HRMS Calcd for C₁₁ H₁₉ NO₅ Cs (M+Cs⁺): 378.0318. Found:378.0318.

E. Allyl 2-acetamido-2-deoxy-3-O-methoxycarbonyl-β-D-glucopyranoside,Compound 1k

The synthesis of Compound 1k was accomplished by the scheme outlinedbelow. ##STR27##

p-Methoxy benzaldehyde (30 mL) was treated with 5 g of dry ZnCl₂. Afterstirring for one hour, Compound 17 (4 g, 15 mmol) was added to themixture at room temperature. The reaction mixture was stirred for 16hours and then treated with 100 mL of CHCl₃ and 100 mL of water. Thecombined organic solutions were washed with 100 mL of brine and dryingover MgSO₄. The solvent was removed in vacuo. The product wasrecrystallized from ethyl acetate/hexane to yield 4.2 g (74 percent) ofallyl2-acetamido-2-deoxy-4,6-O-p-methoxybenzylidene-β-D-glucopyranoside,Compound 18.

¹ H NMR (d-5 pyridine) δ2.08 (s, 3H NHAc), 3.67 (s, 3H, OCH₃), 3.70(ddd, J=9 Hz, J=4 Hz, J=2 Hz, 1H, 5-H), 3.98-4.03 (m, 2H, 6-H₂), 4.27(ddd, J=12.5 Hz, J=5 Hz, J=1 Hz, 1H, CH_(2a) a--C═C), 4.46 (dd, J=12 Hz,J=4 Hz, 1H, CH_(2b) --C═C), 4.52-4.70 (m, 3H, 2-H, 3-H, 4-H), 5.13 (d,J=8.5 Hz, 1H, 1-H), 5.18 (dd, J=9 Hz, J=1 Hz, 1H, CH_(2a) C═C), 5.48(dd, J =17 Hz, J=2 Hz, 1H, CH_(2b) ═C), 5.77 (S, 1H, benzylidene),5.98-6.14 (m, 1H, CH═C), 6.95-7.60 (m, 4H, CH, ar), 9.10 (d, J=8 Hz, 1H,NH).

Compound 18 (400 mg, 1.06 mmol) was dissolved in 3 mL of pyridine. Atzero degrees C, 380 mg (4 equivalents) of methyl chloroformate wereadded. After stirring for 14 hours, the reaction was quenched with icewater. The precipitate was filtered off and washed extensively withwater and ether. The remaining solid was dried and used without furtherpurification 1 to yield 395 mg of allyl2-acetamido-2-deoxy-3-O-methoxycarbonyl-4,6-O-p-methoxybenzylidene-β-D-glucopyranoside,Compound 19a (86.5 percent).

¹ H NMR (d-5 pyridine ) δ2.12 (s, 3H, NHAc), 3.60, (s, 3H, COOCH₃),3.66-3.80 (m, 1H, 5-H), 3.85-4.08 (m, 2H, 6-H₂), 4.23-4.47 (m, 2H, CH₂═--C═C), 4.45-4.55 (m, 1H, 4-H), 4.62 (ddd, J=9 Hz, J=8 Hz, 1H, 1-H),5.15-5.50 (m, 2H, CH₂ ═C), 5.20 (d, J=8 Hz, 1H, 1-H), 5.70 (s, 1H,benzylidene), 5.84 (dd, J=9.5 Hz, J=9.5 Hz, 1H, 3-H), 5.95 (m, 1H,CH═C), 6.90-7.6-(m, 4H, CH, Ar), 9.40 (d, J=8 Hz, 1H, NH).

Compound 19a (0.5 mmol) was treated with 2 mL of glacial acetic acid andstirred for 5 hours at 60° C. The acetic acid was removed in vacuo andthe crude product, Compound 1k, was purified by column chromatography(silica gel, chloroform/MeOH/hexane=6:1:1).

¹ H NMR (D₂ O) δ1.82 (s, 3H, NHAc), 3.38 (ddd, J=10 Hz, J=4.5 Hz, J=2Hz, 1H, 5-H), 3.53 (dd, J=9.5 Hz, J=9 Hz, 1H, 2-H), 3.56-3.82 (m, 3H,4-H, 6-H₂), 4.02 (dd, J=13 Hz, J=6 Hz, 1H, CH_(2a) --C═C), 4.20 (dd,J=13 Hz, J=5 Hz, 1H, CH2b--C═C), 4.57 (d, J=8 Hz, 1H, 1-H), 4.66 (dd,J=10 Hz, J=9.5 Hz, 1H, 3-H), 5.07-5.22 (m, 2H, CH₂ ═C), 5.62-5.83 (m,1H, CH═C).

Yield: 112 mg, 70 percent.

F. Allyl 2-acetamido-3-O-allyloxycarbonyl-2-deoxy-β-D-glucopyranoside,Compound 1l

The synthesis of Compound 11 was accomplished by a similar scheme tothat outlined above in Example 5E.

Starting with 400 mg of Compound 18 and 480 mg of allyl chloroformate,allyl2-acetamido-3-O-allyloxycarbonyl-2-deoxy-4,6-O-p-methoxybenzylidene-β-D-glucopyranoside,Compound 19b was obtained. Yield: 347 mg, 71 percent.

¹ H NMR (d-5 pyridine) δ2.16 (s, 3H, NHAc), 3.65 (s, 3H, OCH₃),3.65-3.75 (m, 1H, 5-H), 3.84-3.94 (m, 2H, 4.20-4.65 (m, 6H, 2-H, 4-H,2CH₂ --C═C), 4.95-5.49 (m, 5H, 2CH₂ ═C, 3-H), 5.22 (d, J=8 Hz, 1H, 1-H),5.72 (s, 1H, benzylidene), 5.95-6.13 (m, 2H, 2CH═C), 7.00-7.70 (m, 4H,CH, Ar), 9.20 (d, J=8 Hz, 1H, NH).

Compound 19b (0.5 mmol) was treated with 2 mL of glacial acetic acid andstirred for five hours at 60° C. The acetic acid was removed in vacuoand the crude product Compound 1l, was purified by column chromatography(silica gel, chloroform/MeOH/hexane=6:1:1).

1H NMR (D₂ O) δ1.76 (s, 3H, NHAc), 3.28-3.38 (m, 1H, 5-H), 3.42-3.76 (m,4H, 2-H, 4-H, 6-H₂), 3.96 (dd, J=13 Hz, J=6 Hz, 1H, CH_(2a) --C═C), 4.14(dd, J=13 Hz, J=5 Hz, 1H, CH_(2b) --C═C), 4.50 (d, J =8 Hz, 1H, 1-H),4.35-4.70 (m, 2H, CH₂ --C═C, alloc), 5.0-5.18 (m, 5H, 2CH₂ ═C, 3-H),5.60-5.84 (m, 2H, CH═C). Yield: 108 mg, 66 percent.

G. Allyl 2-acetamido-2-deoxy-3-O-methoxymethyl-β-D-glucopyranoside,Compound 1m

The synthesis of Compound 1m was accomplished by a similar scheme tothat outlined above in Example 5E.

To a solution of Compound 18 (400 mg, 1.05 mmol) in 10 mL of THF wereadded 50 mg of NaH (60 percent in oil) at zero degrees C. After stirringfor one hour, the reaction mixture was treated with 170 mg ofchloromethylmethyl ether. The reaction was complete after 16 hours, andwas quenched with water. The precipitate of allyl2-acetamido-2-deoxy-3-O-methoxymethyl-4,6-O-p-methoxybenzylidene-β-D-glucopyranoside, Compound 19c, wasfiltered off and washed with water and ether, and was used withoutfurther purification. Yield: 285 mg, 64 percent.

¹ H NMR (d-5 pyridine) δ2.16 (s, 3H, NHAc), 3.45 (s, 3H, OCH₃), 3.65 (s,3H, ar-C-OCH₃, 3.65 (s, 3H, ar--C--OCH₃), 355-3.70 (m, 1H, 5-H),3.80-3.94 (m, 2H, 6H₂), 4.18-4.55 (m, 4H, 2-H, 4-H, CH₂ --C═C),4.80-5.45 (m, 6H, CH₂ ═C, OCH₂ O, 1-H, 3-H), 5.66 (s, 1H, benzylidene),5.92-6.10 (m, 1H, CH═C), 6.90-7.65 (m, 4H, CH, Ar), 9.25 (d, J=8 Hz, 1H,NH).

Compound 19c (0.5 mmol) was treated with 2 mL of glacial acetic acid andstirred for five hours at 60° C. The acetic acid was removed in vacuoand the crude Compound 1m product was purified by column chromatography(silica gel, chloroform/MeOH/hexane=6:1:1).

¹ H NMR (D₂ O) δ1.80 (s, 3H, NHAc), 3.14 (s, 3H, OCH₃), 3.20-3.70 (m,4H, 3-H, 4-H, 6-H₂), 3.92 (dd, J=13 Hz, J=6 Hz, 1H, CH_(2a) --C═C), 4.10(dd, J=13 Hz, J=5 Hz, 1H, CH_(2b) --C═C), 4.35 (d, J=8 Hz, 1H, 1-H),4.50 (d, J =7.5 Hz, 1H, OCH_(2a) O), 4.60 (d, J=7.5 Hz, 1H, OCH_(2b) O),4.98-5.14 (m, 2H, CH₂ ═C), 5.57-5.74 (m, 1H, CH═C). Yield: 110 mg, 72percent.

H. 2-Acetamido-2-deoxy-D-allopyranose,

Compound 1o

The synthesis of Compound 1o was accomplished by the scheme outlinedbelow. ##STR28##

A solution of allyl 2-acetamido-2-deoxy-α-D-glucopyranoside, Compound23, (2.95 g, 11.3 mmol), 2,2-dimethoxypropane (2,.35 g, 22.6 mmol; 2.78mL) and p-toluenesulfonic acid monohydrate (172 mg, 0.90 mmol) inacetone (80 mL) was stirred for two days at room temperature. Duringthis time, another amount of 2,2-dimethoxypropane (2.35 g, 22.6 mmol;2.78 mL) was added to the mixture. After the addition of Et₃ N (1 mL),the mixture was concentrated in vacuo. The residue was chromatographedon silica gel, with toluene-EtOAc (1:2 1:3) to give allyl2-acetamido-2-deoxy-4,6-O-isopropylidene-α-D-glucopyranoside, Compound24 (2.26 g, 66 percent); mp 108.5°-109.0° C. (from EtOAc-hexane).

¹ H NMR (CDCl₃) δ1.44, 1.53 (3H, s, CH₃), 2.04 (3H, s, NHAc), 4.84 (1H,d, J=3.78 Hz, H-1); ¹³ C NMR (CDCl₃) δ19.01, 23.24, 29.01, 54.00, 62.14,63.44, 68.34, 70.56, 74.62, 96.94, 99.87, 118.05, 133.29, 171.37; HRMSCalcd for C₁₄ H₂₃ NO₆ (M⁺): 434.0580. Found: 434.0600.

A mixture of Compound 24 (1.0 g, 3.32 mmol) and Ac₂ O (5 mL) in DMSO (10mL) was stirred for 10 hours at room temperature, and poured intoice-cold aqueous NaOAc. The mixture was stirred for three hours, andextracted with CHCl₃. The extracts were successively washed with aqueousNaHCO₃ and water, dried over anhydrous MgSO₄, and-concentrated. NaBH₄(380 mg, 10.1 mmol) was added to a cooled solution of the residue in CH₂Cl₂ (10 mL), EtOH (10 mL), and water (2 mL) at 0°-5° C., and the mixturewas stirred for 20 minutes at 0°-5° C. To the mixture were added acetone(5 mL) and saturated. NH₄ Cl (5 mL), and the mixture was stirred for 10minutes. The mixture was concentrated and the residue was dissolved inCHCl₃ and water, and the aqueous layer was extracted with CHCl₃. Theextracts were washed with water, dried over anhydrous MgSO₄, andconcentrated. The residue was chromatographed on silica gel, withtoluene-EtOAc (1:3), to give allyl2-acetamido-2-deoxy-4,6-O-isopropylidene-α-D-allopyranoside, Compound25, (612 mg, 61 percent); mp 113.5°-114.5° C. (from EtOAc-hexane).

¹ H NMR (CDCl₃) δ1.45, 1.52 (3H, s, CH₃), 2.04 (3H, s, NHAc), 2.78 (1H,d, J=6.78 Hz, OH), 3.68 (1 H, dd, J=2.77, 9.70 Hz, H-6a), 3.73-3.84 (1H,m), 3.90-4.04 (4H, m, H-3,4, 6b, allylic), 4.24 (1H, br dt, J=3.52, 8.97Hz, H-2), 4.86 (1H, d, J=3.97 Hz, H-1), 5.21-5.34 (2H, m, vinylic ofallyl), 5.81-5.87 (1 H, m, vinylic of allyl), 6.38 (1H, d, J=9.13 Hz,NH); ¹³ C NMR (CDCl₃) δ19.01, 23.16, 28.95, 49.42, 58.36, 62.33, 68.38,69.06, 71.06, 97.15, 99.62, 118.32, 133.17, 169.66; HRMS Calcd for C₁₄H₂₃ NO₆ Cs (M+Cs⁺): 434. 0580. Found: 434. 0551.

A mixture of Compound 25 (489 mg, 1.62 mmol), PdCl₂ (317 mg, 1.79 mmol),and NaOAc (320 mg, 3.90 mmol) in AcOH (10 mL) and water (0.5 mL) washeated at 80° C. for 10 hours. After cooling, the mixture was filtratedthrough a Celite pad, and the filtrate was concentrated. The residue waschromatographed on silica gel, with CHCl₃ -EtOAc-MeOH (5:2:1), to givethe main product, which was acetylated with Ac₂ O (5 mL) and pyridine (5mL) to afford2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-β-D-allopyranose, Compound 26(142 mg, 23 percent) after recrystallization from EtOAc-hexane; mp170.5°-171.0° C.

¹ H NMR (CDCl₃) δ1.96, 1.98, 2.08, 2.13, 2.18 (3H, s, 4 x OAc, NHAc),4.08-4.27 (3H, m, H-5,6a,6b), 4.48 (1H, dt, J=2.98, 9.20 Hz, H-2), 4.98(1H, dd, J=2.81, 9.87 Hz, H-4), 5.57 (1H, br t, J=2.93 Hz, H-3),5.56-5.61 (1H, br s, NH), 5.89 (1H, d, J=8.72 Hz, H-1); ¹³ C NMR (CDCl₃)δ20.42, 20.71, 20.93, 23.01, 49.39, 61.94, 66.18, 69.62, 70.96, 91.35,169.04, 169.51, 169.71, 169.98, 170.68; HRMS Calcd for C₁₆ H₂₃ NOCs(M+Cs⁺): 522. 0376. Found: 522.0376.

A mixture of Compound 26 (77 mg, 0.20 mmol) and methanolic NaOMe (1 mL;M solution) in MeOH (10 mL) was stirred for three hours at roomtemperature, and was neutralized with Dowex 50W-X8 [H⁺ ] resin. Afterthe resin was removed by filtration, the filtrate was concentrated. Theresidue was triturated with MeOH and Et₂ O to give Compound 1o (40 mg,90 percent) as a fluffy solid (α/β=1:2.8).

¹ H-NMR (D₂ O) δ2.00 (s, NHAc of β-isomer), 2.02 (s, NHAc of α-isomer),4.91 (d, J=8.72 Hz, H-1 of β-isomer), 5.09 (d, J=3.50 Hz, H-1 ofα-isomer); ¹³ C NMR (D₂ O) δ (β-isomer) 54.7, 61.6, 66.9, 70.2, 74.2,92.8, 171.5; HRMS Calcd for C₈ H₁₅ NO₆ Cs (M+Cs⁺): 353. 9954. Found:353. 9975.

I. Methyl 2-acetamido-2-deoxy-D-glucopyran-3-uloside, Compound 1q

The synthesis of Compound 1q was accomplished by the scheme outlinedbelow. ##STR29##

A mixture of Compound 24 (690 mg, 2.3 mmol) and Ac₂ O (5 mL) in DMSO (10mL) was stirred for 10 hours at room temperature, and poured intoice-cold aqueous NaOAc. The mixture was stirred for three hours at roomtemperature, and extracted with CHCl₃. The extracts were successivelywashed with aqueous NaHCO₃ and water, dried over anhydrous MgSO₄, andconcentrated. The residue was chromatographed on silica gel, withtoluene-EtOAc (1:4), to give the product, which was crystallized fromEtOAc-hexane, to give allyl2-acetamido-2-deoxy-4,6-O-isopropylidene-α-D-glucopyran-3-uloside,Compound 27 (343 mg, 50 percent); mp 156.0°-156.5° C.

¹ H NMR (CDC₃) δ1.51, 1.53 (3H, s, CH₃), 2.07 (3H, s, NHAc), 3.92-4.03(4H, H-5, 6a, 6b, allylic), 4.11-4.20 (1H, m, allylic), 4.39-4.51 (1H,m, H-4), 4.95 (1H, ddd, J=1.05, 4.25, 7.98 Hz, H-2), 5.18-5.34 (2H, m,vinylic of allyl), 5.32 (1H, d, J=4.25 Hz, H-1), 5.73-5.91 (1H, m,vinylic of allyl), 6.34 (1H, d, J=7.98 Hz, NH); ¹³ C NMR (CDCl₃) δ18.72,22.98, 28.77, 58.72, 62.65, 66.94, 68.80, 76.15, 100.15, 100.46, 118.32,132.73, 170.04, 196.30; HRMS Calcd for C₁₄ H₂₁ NO₆ Cs (M+Cs⁺): 432.0423.Found: 432.0438.

A mixture of Compound 27 (170 mg, 0.57 mmol), PdCl₂ (121 mg, 0.68 mmol),NaOAc (112 mg, 1.36 mmol) in acetic acid (10 mL) and water (0.5 mL) washeated at 80° C. for 10 hours. After cooling, the mixture was filteredthrough a Celite pad, and the filtrate was concentrated. The residue waschromatographed on silica gel, with CHCl₃ -EtOAc-MeOH (5:2:1), duringthe column chromatography methyl glycoside was formed, to give theproduct, which was dissolved in water and lyophilized to give Compound1q (55 mg, 44 percent).

¹ H NMR (D₂ O) δ2.042 (3H, S, NHAc), 3.36 (3 H, s, OMe), 3.76-3.94 (3H,m, H-5,6a,6b), 4.43 (1H, dd, J=0.95, 9.58 Hz, H-4), 4.90 (1H, dd,J=0.98, 4.12 Hz, H-2), 5.16 (1H, d, J=4.08 Hz, H-1); ¹³ C NMR (D₂ O)δ22.00, 55.68, 59.53, 61.00, 72.49, 75.08, 100.85, 174.83, 204.65; HRMSCalcd for C₉ H₁₅ NO₆ Cs (M+Cs⁺): 365. 9954. Found: 365. 9960.

J. Methyl 2-acetamido-2-deoxy-α-D-allopyranoside, Compound 1s

A solution of methyl2-azido-4,6-O-benzylidene-2-deoxy-α-D-allopyranoside (200 mg, 0.65 mmol)and PPh₃ (208 mg, 0.79 mmol) in CH₂ Cl₂ (10 mL) and water (0.5 mL) wasstirred for four hours at room temperature, and the mixture wasconcentrated in vacuo. Active anhydride (2 mL) was added to a solutionof the residue in MeOH (10 mL) at 0°-5° C. and the mixture was stirredfor two hours at 0°-5° C. After the mixture was concentrated, theresidue was chromatographed on silica gel, with toluene-EtOAc(1:2), togive 4,6-O-benzylidene derivative of Compound 21 (110 mg, 72 percent),which was treated with 80 percent AcOH (10 mL) for 3 hours at 80° C.After the mixture was concentrated and dissolved in Et₂ O and water. Theaqueous layer was washed with Et₂ O, and the aqueous layer wasconcentrated. The residue was triturated with MeOH and Et₂ O to give 1s(50 mg, 45 percent); [α]_(D) +77.9° (c 0.68, H₂ O).

¹ H NMR (D₂ O) δ2.00 (3H, s, NHAc), 3.33 (3H, s, OMe), 3.62 (1H, dd,J=2.64, 9.39 Hz, H-4), 3.69-3.89 (3H, m, H-5,6a,6b), 3.96-4.02 (2H, m,H-2,3), 4.71 (1H, d, J=3.76 Hz, H-1); ¹³ C NMR (D₂ O) δ22.22, 50.29,55.84, 61.19, 66.44, 67.33, 69.63, 98.20, 174.19.

K. (2R)-methyl-(3R,4R,5S)-trihydroxypiperidine;(1,5,6-Trideoxy-1,5-imino-D-glucitol), Compound 103c

A solution of (R)-3-azido-2-hydroxypropanal diethyl acetal (Compound I;480 milligrams (mg), 2.54 millimoles (mmol)) in 10 milliliters (mL) of ahydrogen chloride (HCl; pH 1) buffer solution was stirred at 70° C. forfour hours. Gas chromatography analysis [J&W Scientific DB-5 column (15m×0.522 mm), 40° C. for one minute to 250° C. at 20° C./minute] showedcomplete hydrolysis of the acetal (retention time of starting material6.33 minutes, corresponding aldehyde 2.65 minutes). The solution wasadjusted to pH 7, then DHAP (2 mmol) was added and the solutionreadjusted to pH 7. Rabbit muscle FDP aldolase (400 units) was thenadded, and the solution was stirred slowly for 36 hours. Enzymatic assayshowed no DHAP remaining.

Barium chloride (BaCl₂.2H₂ O) [1.22 grams (g), 4.80 mmol] and twoequivalent volumes of acetone were added to the solution. The solutionwas maintained at -20° C. for about 18 hours. The precipitate wasrecovered, and treated with Dowex X 50(H⁺) in 20 mL water to removebarium cation. After filtration, the solution was adjusted to pH 7 andthen lyophilized to obtain Compound 101c (550 mg, 1.79 mmol, 90 percentbased on DHAP) as a white hygroscopic solid: R_(f) =0.46 percentpalladium/carbon catalyst (Pd/C) under 45 pounds per square inch (psi)of hydrogen (H₂) for 18 hours. The catalyst was removed by filtrationand the filtrate was concentrated and chromatographed on a short silicagel column [chloroform (CHCl₃): methanol (MeOH): H₂ O=5:5:2] to yieldthe title compound, Compound 103c (250 mg, 95 percent) as a white fluffycompound: R_(f) 0.60 (2-propanol: NH₄ OH: H₂ O=6:3:2); [α]_(D) ²³ +12.0°(c 2.5 H₂ O); ¹ H-NMR (D₂ O) δ1.10 (3H, d, J=6.4 Hz, CH₃), 1.27 (3H, d,J=6.8 Hz, 5-epimer-CH₃), 2.48 (1H, t, J_(1a),1e =J_(1a),2 =12 Hz, H-1a),2.63 (1H, dd, J₅,6 =6.4, J₅,4 =3.6 Hz, H-5), 3.03 (1H, t, J₃,4 =J₄,5 =9Hz, H-4), 3.47-3.52 (1H, m, H-2) ppm; ¹³ C-NMR (D₂ O) δ16.82, 48.22,55.76, 69.98, 75.37, 77.83 ppm. HRMS (M+H⁺) calculated 148.1001, found148.0979.

L. (2R)-methyl-(3R,4R,5R)-trihydroxypiperidine;(1,5,6-Trideoxy-1,5-imino-D-mannitol), Compound 103a

A solution of (S)- or (RS)-3-azido-2-hydroxypropanal diethyl acetal (480mg, 2.54 mmol) in 10 mL of the pH 1 buffer solution was stirred at 70°C. for four hours. Gas chromatography analysis (J&W Scientific DB-5column (15 m×0.522 mm), 40° C. for one minute to 250° C. at 20°C./minute) showed complete hydrolysis of the acetal (retention time ofstarting material 6.33 minutes, corresponding aldehyde 2.65 minutes).The solution was adjusted to pH 7, then DHAP (2 mmol) was added and thesolution readjusted to pH 7. Rabbit muscle FDP aldolase (400 units) wasthen added, and the solution was stirred slowly for 36 hours. Enzymaticassay showed no DHAP remaining.

Barium chloride (BaCl₂.12.2H₂ O) (1.22 g, 4.80 mmol) and two equivalentvolume of acetone were added to the solution. The solution wasmaintained at -20° C. for about 18 hours. The precipitate was recovered,and treated with Dowex X 50(H⁺) in 20 mL water to remove barium cation.After filtration, the solution was adjusted to pH 7 and then lyophilizedto obtain the phosphorylated azidoketose.

A solution of this azido α-ketose phosphate in 0 mL of water washydrogenated with 50 mg 10 percent Pd/C under 45 psi of hydrogen for 18hours. The catalyst was removed by filtration and the filtrate wasconcentrated and chromatographed on a short silica gel column (CHCl₃ :MeOH: H₂ O=5:5:2) to yield the title compound, Compound 103a: R_(f) 0.12(CHCl₃ : MeOH: H₂ O=5:1.5); [α]_(D) ²³ -4° (c=2.5, H₂ O).

¹ H-NMR (D₂ O) δ1.213 (3H, d, J=6.5 Hz, CH₃), 2.893 (1H, dd, J₄,5 =9.5Hz, J₅,6 =6.5 Hz, H-5), 3.00 (1H, d, J_(1a),1e =13.5 Hz, H-1a), 3.16(1H, dd, J_(1e), 1a =13.5 Hz, J_(1e), 2 =3 Hz, H-1e), 3.45 (1H, t,J_(1e),2 =J₂,3 =3 Hz, H-2), 3.46 (1H, t, J=9.5 Hz, H-4), 3.675 (1H, dd,J₃,4 =9.5 Hz, J₂,3 =3 Hz, H-3) ppm; ¹³ C-NMR (D₂ O) δ15.24 (CH₃),48.31(C-1), 56.17(C-5), 66.74, 70.88, 72.92 ppm. HRMS (M+H⁺) calculated148.0974, found 148.0900.

M. (2R)-Methyl-(3R,4R)-(5R)-N-acetylpiperidine;(1,5,6-trideoxy-1,5-imino-N-acetylglucosamine; Compound 103b), and(2R)-methyl-(3R,4R)-dihydroxy-(5S)-N-acetylpiperidine;(1,5,6-trideoxy-1,5-imino-N-aceytylmannosamine; Compound 103d

To a mixture containing 100 mL of dichloromethane (CH₂ Cl₂), 5.27 g(36.3 mmol) of (R)-2-(diethoxymethyl)aziridine (Compound II, 95 percentee) and 40.0 g (289.4 mmol) of potassium carbonate (K₂ CO₃) was added4.0 mL (42.4 mmol) of acetic anhydride. The mixture was stirred at roomtemperature for 10 hours, filtered, and the filtrate was removed underreduced pressure. The residue was purified by silica gel columnchromatography to yield 4.27 g of Compound IIIa: 63 percent yield;[α]_(D) ²³ +84.23° (c 1.5, CHCl₃).

¹ H-NMR (CDCl₃) δ1.22, 1.25 (each 3H, t, J=7.0 Hz, CH₂ CH₃), 2.17 (3H,s, CH₃ CO), 2.28 (1H, d, J=3.3 Hz, CH₂ of aziridine) 2.35 (1H, d, J=6Hz, CH₂ of aziridine), 2.68 (1H, m, CH of aziridine), 3.51-3.78 (4H, m,OCH₂), 4.40 (1H, d, J=4.5 Hz, CH(OEt)₂) ppm; ¹³ C-NMR (CDCl₃) δ15.6(2C), 23.8, 27.7, 38.3, 63.2, 63.3, 101.6, 183.2 ppm. HRMS (M+H⁺)calculated 188. 1286, found 188.1290.

Compounds IIIb, IIIc and IIId were similarly prepared using appropriateblocking groups.

To a mixture containing 423.0 mg (2.26 mmol) of Compound IIIa and 1.9 g(29.5 mmol) of sodium azide in 18 mL of dimethyl formamide (DMF) wasadded 18.0 mL of zinc chloride [1.0M solution in ether], and thereaction mixture was stirred at 75° C. for three days. The mixture wasextracted with ethyl acetate (EtOAc) and the organic layer was washedwith water, dried over magnesium sulfate (MgSO₄) and concentrated. Theresidue was purified by silica gel chromatography (hexane: EtOAc=3:2) toyield 318.6 mg of Compound IVa [61 percent yield, [α]_(D) ²³ -23.8° (c0.15, CHCl₃)].

¹ H-NMR (CDCl₃) δ1.23 (6H, t, J=7.1 Hz, CH₂ CH₃), 2.03 (3H, s, CH₃ CO),3.45-3.61 and 3.66-3.76 (6H, m), 4.24 (1H, m, --CHNH), 4.53 (1H, d,J=3.9 Hz, --CH(OEt)₃), 5.83 (1H, d, J=7.8 Hz, --NH) ppm; ¹³ C-NMR(CDCl₃) δ15.5, 15.6, 23.7, 50.8, 51.0, 63.7, 64.4, 101.3, 170.5 ppm.HRMS (M+Cs⁺) calculated 363.0433, found 363.0450.

The aldehyde liberated from racemic Compound IVa (1 g) was mixed with 18mL of DHAP (71.3 mmol), and the pH was adjusted to 6.5 with 1 normal (N)sodium hydroxide (NaOH). To this solution, rabbit muscle FDP aldolase(400 units) was added, and the mixture was stirred slowly for 4.5 hours.The mixture was passed through Dowex 1 (HCO₂ ⁻) and eluted with water(400 mL) 0.1 molar (M) sodium chloride (NaCl; 250 mL), 0.4M NaCl (700mL), and 0.5M NaCl solution, successively.

After adding 200 mL of water to the fraction eluted by the 0.4M NaClsolution (700 mL) that contained Compound 10lb, Pd/C (103.0 mg) wasadded, and the mixture was hydrogenated under the pressure of 50 psi forone day. The catalyst was filtered off and the 10 filtrate waslyophilized. The residue was treated with a mixed solvent [chloroform(CHCl₃): methanol (MeOH): H₂ O=6:4:1]. The soluble portion was collectedand purified by silica gel chromatography (CHCl₃ : MeOH: H₂ O=6:4:0.7)to yield Compounds 103b and 103d in a 12:1 ratio. Starting withenantiomerically pure aldehyde substrates, Compounds 103b and 103d wereseparately obtained.

Compound 103d

¹ H-NMR (D₂ O) δ1.33 (3H, d, J=6.3 Hz, H-6), 1.94 (3H, s, CH₃ CO), 2.85(1H, t, J=12.5 Hz, H-1a), 3.10 (1H, m, H-5), 3.36 (1H, dd, J =12.5 and4.9 Hz, H-1e), 3.39, 3.51 (each 1H, t, J=9.8 Hz, H-3,4), 3.99 (1H, ddd,J=12.5, 9.8 and 4.9 Hz, H-2) ppm; ¹³ C-NMR (D₂ O) δ14.8, 22.3, 44.0,48.2, 54.9, 72.9, 73.1, 174.2 ppm. HRMS (M+Na⁺) calculated 211.1059,found 211.1053.

Compound 103b:

¹ H-NMR (D₂ O) δ1.34 (3H, d, J=6.6 Hz, H-6), 1.97 (3H, s, CH₃ CO), 3.10(1H, m, H-5), 3.15, 3.43 (each 1H,dd, J=13.7 and 3.0 Hz, H-1), 3.62 (1H,t, J=9.4 Hz, H-4), 3.80 (1H, dd, J=9.4 and 4.6 Hz, H-3), 4.32 (1H, dt,J=4.6 and 3.0 Hz, H-2) ppm; ¹³ C-NMR (D₂ O) δ14.5, 22.4, 44.4, 47.6,55.0, 69.9, 70.0, 174.7 ppm. HRMS (M+Na⁺) calculated 211.1059, found211.1050.

N. (1,2R)-dimethyl-(3R,4R,5S)trihydroxypiperidine;(N-Methyl-1,5,6-trideoxy-1,5-imino-D-glucitol), Compound 117

Compound 103c (47 mg, 0.32 mmol), formaldehyde (300 ml, 37 percent byweight solution) and 10 mg of 10 percent Pd/C were hydrogenated under 45psi of hydrogen in 10 mL of MeOH/H20 (1:1) solution for one day. Afterfiltration, the solvent was removed under reduced pressure to yieldCompound 117 (52 mg, quantitative yield) as hygroscopic material: Rf=0.65 (2-propanol: NH₄ OH: H₂ O=6:3:3:2); [a]_(D) ²³ +4.58° (c 1.75, H₂O).

¹ H-NMR (D₂ O) δ1.12 (3H, d, J=6.5 Hz), 2.36 (1H, dd, J=11.5, 6.5 Hz),2.63 (1H, d, J=5 Hz), 3.02-3.06 (2H, m), 3.18 (1H, t, J=9.5 Hz),3.48-3.53 (1H, m) ppm; ¹³ C-NMR (D₂ O) δ16.96, 43.87, 61.17, 65.96,70.68, 76.64, 79.95 ppm. HRMS (M+H⁺) calculated 161.1052, found162.1129.

O. (1,2R)-dimethyl-(3R,4R,5S)-trihydroxypiperidine oxide;(N-Methyl-1,5,6-trideoxy-1,5-imino-D-glucitol oxide),

Compound 118

Hydrogen peroxide (42 mg, 50 percent by weight solution) was added to a1 mL H₂ O solution containing Compound 117 (10 mg, 0.062 mmol) and themixture was stirred at room temperature for three days. The solvent wasremoved under reduced pressure to obtain pure Compound 118 (10 mg, 91percent) as a single stereoisomer of white hygroscopic compound: R_(f)=0.53 (2-propanol: NH₄ OH: H₂ O=6:3:2); [a]_(D) ²³ +5.40° (C 3.00, H₂O).

¹ H-NMR (D₂ O) δ1.12 (3H, d, J=6.5 Hz, CH₃), 3.14 (1H, dd, J₅,4 =10,^(J) 5, CH₃ =6.5 Hz, H-5), 3.20 (1H, t, J₂.3 =J₃.4 =10 Hz, H-3), 3.28(1H, t, J_(1a),1e =J_(1a),2 =10 Hz, H-1a), 3.39 (1H, dd, J_(1e),1a =10,J_(1e),2 =5 Hz, H-1e), 3.41 (1H, t, J₃,4 =J₄,5 =10 Hz, H-4), 3.88 (1H,td, J_(1a),2 =J₂₃ =10, J₂,1e =5 Hz, H-2) ppm; ¹³ C-NMR (D₂ O) δ8.65,55.89, 67.85, 64.52, 70.21, 70.60, 75.44 ppm. HRMS (M+H⁺) calculated177.2009, found 177.2014.

P. (2S)-methyl-(3S, 4S, 5S)-trihydroxypiperidine;[1,6-L-rhamnanojirimycin (rhamnojirimycin)], Compound 106

To an aqueous solution of (RS) or (R)-3-azido-2-hydroxypropanal,prepared by heating a suspension of 3-azido-2-hydroxypropanal diethylacetal (1.1 g, 5.8 mmol) in pH 1.0 buffer (40 mL) at 45° C. for 12hours, were added DHAP (1.9 mmol) and Tris buffer (675 mM, KCl 750 mM,pH=7.5; 5.0 mL), and the pH value of the resulting solution was adjustedto 7.5 with 1N NaOH. To prepare a source for rhamnulose-1-phosphatealdolase, E. coli strain K-40 was treated with lysozyme (from egg white;10 mg) in Tris buffer (45 mM, potassium chloride (KCl) 50 mM, pH=7.5; 20mL) for one hour at 35° C. One gram of this E. coli preparation wasadded to the above pH-adjusted solution, and the mixture was stirredslowly until 90 percent of DHAP was consumed.

After the reaction, the solution was adjusted to pH 7.0, BaCl₂.2H₂ O(950 mg, 3.9 mmol) was added, and the resulting precipitate was removedby centrifugation. Acetone (twice the volume) was added to thesupernatant. The mixture was kept in a refrigerator for two hours andthe precipitate newly appeared was collected. To remove the barium ion,Dowex 50 (H⁺) was added with stirring followed by filtration. Thesolution was lyophilized and the residue was purified by silica gelchromatography (CHC₃ :MEOH: H₂ O=8:2:0.1) to yield the phosphorylatedazidoketose, Compound 104.

Compound 104 in ethanol (30 mL) containing Pd/C (20 mg) was hydrogenatedat 50 psi for one day. The catalyst was filtered off and the filtratewas concentrated. The residue was purified by silica gel chromatography(CHCl₃ : MeOH: H₂ O=6:4:1˜5:5:2) to yield Compound 106.

Compound 104a (dephosphorylated): Yield-55 percent (based on DHAP), ¹³C-NMR (CD₃ OD) δ54.6, 64.2, 76.8, 77.6, 81.1, 103.3 ppm.

Compound 106: ¹ H-NMR (D₂ O) δ1.00 (3H, d, J=6.5, 5-CH₃), 2.30 (1H, m,H-5), 2.56 (1H, d, J=14.4, H-1a), 2.78 (1H, dd, J=14.4, 2.3, H-1e), 3.14(1H, t, J=9.9, H-4), 3.35 (1H, dd, J=9.9, 2.9, H-3), 3.82 91H, bs, H-2)ppm; ¹³ C-NMR (D₂ O) δ17.4, 48.6, 55.6, 69.8, 74.3, 74.5 ppm. HRMS(M+Cs⁺) calculated 279.9950, found 279.9950.

Q. (2R)-Methyl-(3S,4R,5S)-trihydroxypiperidine;(D-1,6-D-dideoxygalactojirimycin), and(2S)-methyl-(3S,4R,5S)-trihydroxypiperidine;(L-1,6-dideoxyaltrojirimycin), Compounds 110 and 109

To an aqueous solution of (RS)- or (R)-3-azido-2-hydroxypropanal,prepared by heating a suspension of 3-azido-2-hydroxypropanal diethylacetal (1.1g, 5.8 mmol) in pH 1.0 buffer (40 mL) at 45° C. for 12 hours,were added DHAP (1.9 mmol) and Tris buffer (675 mM, KCl 750 mM, pH=7.5;5.0 mL), and the pH was adjusted to 7.5 with 1N NaOH. To prepare asource for fuculose-1-phosphate aldolase, E. coli strain K-58 wastreated with lysozyme (from egg white; 10 mg) in Tris buffer (45 mM,potassium chloride (KCl) 50 mM, pH=7.5; 20 mL) for one hour at 35° C.One gram of this E. coli preparation was added to the above pH-adjustedsolution, and the mixture was stirred slowly until 90 percent of DHAPwas consumed. E. coli fuculose-1-phosphate aldolase has been cloned andoverexpressed, providing an alternate source for the enzyme, [Ozaki etal., J. Am. Chem. Soc., 112::4970 (1990)].

After the reaction, the solution was adjusted to pH 7.0, BaCl₂.2H₂ O(950 mg, 3.9 mmol) was added, and the resulting precipitate was removedby centrifugation. Acetone (twice the volume) was added to thesupernatant. The mixture was kept in a refrigerator for two hours andthe precipitate newly appeared was collected. To remove the barium ion,Dowex 50 (H⁺) was added with stirring followed by filtration. Thesolution was lyophilized and the residue was purified by silica gelchromatography (CHCl₃ : MEOH: H₂ O=8:2:0.1) to yield a phosphorylatedazidoketose, Compound 108, in 20 percent (based on DHAP). Compound 108:¹³ C-NMR (CD₃ OD) δ52.7, 66.7, 71.9, 72.8, 80.1, 104.4 ppm.

A solution of Compound 108 in ethanol (30 mL) containing Pd/C (20 mg)was hydrogenated at 50 psi for one day. The catalyst was filtered offand the filtrate was concentrated. The residue was purified by silicagel chromatography (CHCl₃ : MeOH: H₂ O=6:4:1˜5:5:2) to yield anapproximately equimolar mixture of Compounds 109 and 110.

Compound 110: [α]_(D) +18.2° (c 1.1, MeOH), ¹ H-NMR (D₂ O) δ1.20 (3H, d,J=6.7, 5-CH₃), 2.71 (1H, t, J=12.0, H-1a), 3.30 (1H, qd, J=6.7, 1.5,H-5), 3.31 (1H, dd, J=12.0, 5.5, H-1e), 3.50 (1H, dd, J=9.7, 3.0, H-3),3.87 (1H, dd, J=3.0, 1.5, H-4), 3.90 (1H, ddd, J=11.5, 9.5, 5.5 Hz, H-2)ppm; ¹³ C-NMR (D₂ O) δ14.4, 46.5, 55.3, 64.8, 70.3, 73.5 ppm. HRMS(M+H⁺) calculated 148.0974, found 148.0974.

R. (2R)-Methyl-5-fluoro-(3R,4R,5R)-trihydroxypiperidine;(2,6-Dideoxy-2-fluoromannojirimycin), Compound 103e

To a stirring solution of 3-azido-2-hydroxypropanal diethyl acetal (7.32g, 38.73 mmol) in dry benzene (50 mL) was addeddiethylaminosulfurtrifluoride (DAST; 20.6 mL) at -78° C. After theaddition, the solution was stirred at room temperature for an hour, thenheated to 70° C. for 12 hours. The reaction was quenched by the additionof methanol at zero degrees C and diluted with water. Afterdichloromethane extraction, the organic layer was dried over MgSO₄ andconcentrated in vacuo. The crude product was purified with silica gelcolumn chromatography (hexane: ether=9:1, volume/volume) to yield3-azido-2-fluoropropanal diethyl acetal as an oil (65 percent); R_(f)=0.84 EtOAc: hexane=2:3).

¹ H-NMR (CD₃ Cl) δ1.215˜1.219 (6H, m) 3.526 (2H, din, J=15.3 Hz),3.642˜3.670 (2H, m), 3.680˜3.808 (2H, m), 4.514 (1H, din, J=45.9 Hz)ppm.

A mixture of racemic 3-azido-2-fluoropropanal diethyl acetal (750 mg,3.93 mmol) and 1N HCl (20 mL) was heated at 65° C. for 30 hours. Themixture was cooled to room temperature and DHAP (1 mmol) was added, andthe pH value was adjusted to 7 with 10N NaOH. Rabbit muscle FDP aldolase(500 Units) was added to the pH-adjusted solution and the resultingsolution was stirred slowly for 36 hours. Enzymatic determinationindicated that all of the DHAP had been consumed. The solution was thenfiltered and lyophilized. The yellow syrup was treated with methanol andfiltered to remove the insoluble material. The methanol was removedunder reduced pressure to provide Compound 101e.

A solution containing this product (20 mg) and 10 percent Pd/C (5 mg) in10 mL methanol was hydrogenated at 50 psi for one day. The catalyst wasfiltered off and the solvent was removed under reduced pressure. Thecrude product was purified by silica gel column chromatography (CHCl₃ :MeOH=3:1) to yield 2,6-dideoxy-2-fluormannojirimycin, Compound 103e.

S. 1,6-Dideoxyidojirimycin; Compound Compound 111 is prepared in amanner similar to that used for the preparation of Compound 106 (Example5) except that (S)-3-azido-2-hydroxypropanal is utilized with DHAP andrhamnulose-1-phosphate aldolase.

T. (3S,4S)-Dihydroxypiperidine, Compound 114a;(3R,4R)-dihydroxy-(6R)-methypiperidine, Compound 114b;(3S,4S)-dihydroxy-(5R)-methypiperidine, Compound 114c

Compound 114a was produced by the DERA-catalyzed condensation of(RS)3-azido-2-hydroxypropanal, prepared as above, and acetaldehyde.Resulting Compound 113a was recovered and hydrogenated over Pd/C asdescribed before to provide Compound 114a.

Compound 114a:

¹ H-NMR (D₂ O) δ1.51 (2H, m, H-2), 2.55 (1H, ddd, J=13.1, 7.6, 4.8,H-1), 2.67 (1H, dd, J=13.4, 3.0, H-5), 2.90 (1H, dd, J=13.4, 5.7, H-5),2.86-2.96 (1H, m, H-1), 3.67 (1H, dt, J=5.9, 2.5, H-4), 3.74 (1H, ddd,J=7.6, 4.6, 3.0 H-3)ppm; ¹³ C-NMR (D₂ O) δ29.9 (C-2), 41.9 (C-1), 48.1(C-5), 68.8, 69.3 (C-3, C-4) ppm. HRMS (M⁺): Calculated: 117.0790, found117.0785.

Compound 114a was produced by the DERA-catalyzed condensation of(RS)3-azido-2-hydroxypropanal, prepared as above, and acetone. ResultingCompound 113b was recovered and hydrogenated over Pd/C as describedabove to provide Compound 114b.

Compound 114b:

¹ H-NMR (CDCl₃) δ1.05 (3H, d, J=6.3, H-1), 1.27 (1H, q, J=12.4, H-3a),1.67 (1H, ddd, J=12.5, 4.7, 2.5, H-3e), 2.55 (1H, ddq, 12.6, 6.3, 2.5,H-2), 2.62 (1H, dd, J=13.4, 1.3,-H-6a), 3.06 (1H, dd, J=13.4, 2.9,H-6e), 3.25 (3H, br-s, 2 OH, NH), 3.53 (1H, ddd, J=11.9, 4.7, 3.0, H-4),3.69 (1H, br s, H-5) ppm; ¹³ C-NMR (CDCl₃) δ22.1 (C-1), 37.7 (C-3),50.1, 50.5 (C-2, C-6), 67.2 69.9 (C-4, C-5) ppm. HRMS (M+Cs⁺):Calculated 264.0001, found 264.0000.

Compound 114c was produced by the DERA-catalyzed condensation of (RS)3-azido-2-hydroxypropanal, prepared as above and propionaldehyde.Resulting Compound 113o was recovered and hydrogenated over Pd/C asdescribed above to provide Compound 114c.

Compound 114c

¹ H-NMR (D₂ O) δ0.91 (3H, d, J=7.0, CH₃), 1.77-1.82 (1H, m, H-2), 2.45(1H, t, J=12.4, H-1a), 2.67 (1H, t, J=11.7, H-5a), 2.70 (1H, dd, J=12.4,4.8, H-1e), 2.90 (1H, dd, J=11.9, 4.6, H-5e), 3.72 (1H, ddd, J=11.7,5.1, 3.0, H-4), 3.85 (1H, br s, H-3) ppm; ¹³ C-NMR (D₂ O) δ15.4 (CH₃),35.5 (C-2), 44.8, 45.7 (C-1, C-5), 67.0, 72.6 (C-3, C-4) ppm. HRMS(M+Cs⁺) Calculated 264.0001, found 264.0003.

Example 6

Inhibition Studies

A. Inhibition Study

Materials

All of the buffers, enzymes, and substrates were purchased from Sigmaand used as received. These includedpiperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), sodium acetate(NaOAc), ethylenediaminetetraacetic acid (EDTA), β-D-glucosidase (fromsweet almond), p-nitrophenyl β-D-glucoside, β-nitrophenylα-D-glucosidase, p-nitrophenyl α-D-glucoside,β-N-acetyl-D-glucosaminedase, p-nitrophenyl β-N-acetyl-D-glucosaminide,α-D-mannosidase and p-nitrophenyl α-D-mannoside.

B. Preparation of solutions:

(a) PIPES buffer (0.05M with 0.01 mM EDTA, pH 6.5): To 1 liter (L)deionized H₂ O were added 15.1 g PIPES and 35.7 mg EDTA. The pH wasadjusted to 6.5 with NaOH (10M).

(b) PIPES-NaOAc buffer (0.01M PIPES, 0.2M NaOAc and 0.01 mM EDTA, pH6.5). This buffer was prepared according to the literature procedure[Dale et al., Biochemistry, 24:3530 (1985)].

(c) β-D-Glucosidase: The stock enzyme solution was prepared bydissolving 15 mg of solid protein (4 units/mg) in 1 mL PIPES-NaOAcbuffer solution. This stock enzyme solution was diluted 5-fold for theenzymatic assay.

(d) α-D-Glucosidase: 1.5 mg of solid protein (70 units/rag) weredissolved in 1 mL PIPES-NaOAc buffer solution and used for assayswithout further dilution.

(e) β-N-Acetyl-D-Glucosaminidase: 25 units of the protein were suspendedin 0.55 mL of 3.2M ammonium sulfate [(NH₄)₂ SO₄ ] solution asdistributed by Sigma.

(f) α-D-Mannosidase: 5 mg of the solid protein were suspended in 1 mL of3.0M (NH₄)₂ SO₄ and 0.1 zinc acetate (ZnOAc), as distributed by Sigma.

(g) Substrate solutions: all substrates were dissolved in thecorresponding buffer solution for enzymatic assay.

C. General Procedure for Enzyme Assay

For each inhibitor, five inhibitor concentrations, ranging from zero tothree times K_(i), were generally used to determine the K_(i) value. Ateach inhibitor concentration, six substrate concentrations, spanningfrom 0.4 K_(m) to 4 K_(m), were used to obtain a single Lineweaver-Burkplot. The amount of enzyme added in each assay was adjusted so that lessthan 10 percent of the substrate, with its lowest substrateconcentration, would be consumed within 45 seconds. Since all of thesubstrates have p-nitrophenol as leaving group, the assays weremonitoredat 400 nanometers (nm), where the molecular extinctioncoefficient, ε, was calibrated to be 3204.5M⁻¹ cm⁻¹ at pH 6.5. Thefollowing illustrates the detailed procedure.

To a 1 mL disposable cuvette were added 950 microliters (μL) of theNaOAc-PIPES buffer solution, 20 μL of the inhibitor solution and 20 μLof the p-nitrophenyl β-D-glucoside solution (100 mM in PIPES-NaOAcbuffer, pH 6.5). The solution was well mixed and 20 μL of theβ-D-glucosidase solution were injected into the cuvette to start thereaction. The reaction was monitored at 400 nm on a Beckman DU-70photospectrometer for 45 seconds and the initial hydrolysis rate wascalculated. The same procedure was repeated with five other substrateconcentrations. After all the initial rates were accumulated, thecorresponding Lineweaver-Burk plot at that inhibitor concentration wasconstructed.

PIPES-NaOAc buffer was used for all the enzymes exceptβ-N-acetyl-D-glucosaminidase, for which PIPES buffer was used.

Exemplary K_(i) data are provided in the Table 3, above.

Example 7

Expression of Gait in E. coli Strain

JM109

The plasmid pIN-GT (See FIG. 1) in E. coli strain SB221 was isolated andtransformed into strain JM109 (ATCC 53323) using a well known protocol.The transformants were introduced directly into 150 mL LB media andgrown without induction overnight at 37° C. Cells were harvested bycentrifugation at 4,000×g at 4° C. The cells were resuspended in 3 mL LBmedia and 1 mL chloroform was added. The mixture was allowed to stand atroom temperature for 15 minutes before addition of 30 mL of 50 mM HEPESbuffer, pH 7.4. Similarly, SB221 was freshly transformed and grown as acontrol. The enzyme was isolated and the activity was determinedaccording to reported procedures.

Example 8

Studies with Focosyltransferas

Further studies of synthesis and inhibition using UDP-fucose as donor,fucosyltransferase and N-acetyllactosamine (Galβ,4GlcNAc) as acceptorwere carried out, as were inhibition studies with oligosaccharides.Those studies are discussed below.

A. Results

The K_(m) for N-acetyllactosamine at 0.2 mM GDP-fucose was determined toby 6±3 mM. Galactose β1,4-glucal (Table 1, from Compound 6) and3-deoxy-N-acetyllactosamine (Table 1, Compound 1y) did not exhibit anyinhibitory effect up to concentrations of 50 mg/mL under the describedconditions. Galactose β1,4-5-thioglucose (Table 1, from Compound 5)proved to be a good substrate for fucosyltransferase in that a reactionwith 5 mM of this compound reacted 2-5 times faster thanN-acetyllactosamine. Galactose β1,4-deoxynojirimycin (Table 1, Compound4) was an inhibitor of fucosyltransferase with an IC₅₀ approximatelyequal to 40 mM. During the course of these studies, it was noted thatGDP-fucose exhibits very strong substrate inhibition against thesynthesis of the trisaccharide.

Assay for Fucosyltransferase

The assay procedure for determining fucosyltransferase activity wasessentially as described by Lowe et al., Genes and Development, 4:1288(1990); Lowe et al, Cell, 63:475 (1990) with some minor modifications.

For some studies, a stock solution (mix A) containing 1 mM GDP-fucose,67500 cpm ¹⁴ C-GDP, fucose (Amersham Corp., 290 mCi/mmol), 25 mM ATP, 50mM fucose, and 250 mM sodium cacodylate buffer, pH 6.2 was mixed freshthe day of use and stored on ice.

A second set of solutions (mixes B1-B6) contained N-acetyllactosamine invaried concentrations from 1.5 to 50 mM and 100 mM MnCl₂. Thesesolutions were also made fresh each day and stored on ice. Assaysproceeded by mixing 2 μL of mix A with 2 μL of one of the B mixes. Tothis solution, 5 μL of water were added followed by initiation of thereaction by addition of 1 μL enzyme solution. This assay mixture wasgently mixed and allowed to incubate at 37° C. for 30 minutes.

For other studies, such as those using a recombinantfucosylα1,3/4transferase (EC 2.4.1.65) whose results are shown in Table1a, a stock mixture containing 0.25 mM ¹⁴ C-GDP-fucose (5000 cpm/μL),6.25 mM ATP, 25 mM MnC₂ are 62.5 mM cacodylate buffer, pH 6.2, wasadmixed fresh on the day of use, and stored on ice. To that solution,Fuc T was added immediately before use, and the reaction was initiatedby the combination of 16 μL of that mixture with 4 μL of 100 mM acceptorsubstrate. The resulting admixture was then incubated at 37° C. for30-240 minutes, depending upon the acceptor substrate (reactantcompound) under study.

Simultaneous to these assays, another assay was performed and handledidentically in the absence of lactosamine for determination of thebackground radioactivity either inherent in the study or from thegeneration of ¹⁴ C-fucose by the action of some contaminatingphosphatase.

Upon completion of the incubation, 400 μL of a 20 percent (v/v)suspension of QAE-Sephadex was added. These suspensions were gentlymixed at room temperature for 10 minutes before centrifugation at 13,000rpm for one minute. From the supernatant fluid, 100 μL were extractedand mixed with 10 mL of scintillation cocktail. The radioactive contentwas measured on a Beckmann LS1701 scintillation counter. Care was takento insure that less than 10 percent of the enzymatic reaction had takenplace over the 30 minute incubation period.

The Michaelis constant (K_(m)) for lactosamine in the presence of 0.2 mMGDP-fucose was determined by fitting the data to equation 1 by nonlinearregression analysis.

    v=(v.sub.max)S/(K.sub.m +S)                                equation 1

In the above equation, v =reaction rate, V_(max) =maximal velocity, andS=N-acetyllactosamine concentration.

Inhibition studies were carried out in an analogous manner in thepresence of 2 mM N-acetyllactosamine and varied concentrations from 1 to50 mg/mL of (a) galactosylβ1,4glucal (Compound 8), (b)galactosylβ1,4deoxynojirimycin (Compound 10a, (c)galactosylβ1,4-5-thioglucose (Compound 7), and (d)3-deoxy-N-acetyllactosamine (Compound 2i). Percent inhibition wascalculated as the fraction of inhibited activity to the uninhibitedreaction rate. These fractions were plotted verus inhibitorconcentration. The data was fit to a straight line (equation 2) bylinear regression, and 50 percent inhibitory concentration (IC₅₀) wasextrapolated from this line as the inhibitor concentration that wouldgive 50 percent inhibition of the fucosyltransferase reaction. In thisequation, m=slope and b=y-intercept.

    Percent inhibition=m([inhibitor])+b                        equation 2

The results of these assays are shown in Table 1a.

Example 9

Comparison of Native and Modified CMP-sialic acid synthetase enzymes

A. Preparation of enzymes

1. Construction of plasmids for the native and modified CMP-N-sialicacid synthetase enzymes

The 1.3 kb NeuAc gene coding for the native CMP-sialic acid synthetasewas amplified by PCR using the primers shown below as SEQ ID NO's: 4 and5 with pWA1 plasmid DNA as a template. Innis et al., PCR Protocols, Aguide to methods and applications; Academic Press, San Diego, Calif.(1990). ##STR30##

The PCR product was purified by phenol extraction followed by gelfiltration on a Bio Gel P-10 spin columns in TE buffer. The purifiedoligonucleotide was digested with Eco RI and Hind III and purified by anagarose gel electrophoresis in low melting point agarose. This fragmentwas then ligated into plasmid pKK 223-3 under the control of the tacpromotor. Zapata et al., J. Biol. Chem., 264:14769 (1989) and-Tabor etal., Proc. Natl. Acad. Sci. USA, 82:1074 (1985). This plasmid wasdesignated pWG123. Plasmid pWG123 was then transformed into E. coli Surestrain obtained from Stratagene Co.

The construction of plasmid CMPSIL-1, which contains the modifiedCMP-NeuAc synthetase gene (Example 2), was accomplished by the method ofIchikawa et al., J. Am. Chem. Soc., 113:4698 (1991). The CMP-NeuAcsynthetase gene was fused with the decapeptide tag sequenceTyr-Pre-Tyr-Asp-Val-Pro-Asp-Tyr-Ala-Ser (SEQ ID NO:3) at the C-terminus,cloned into a lambda ZAP™ vector at EcoRI and XbaI sites andoverexpressed in E. coli sure strain.

To subclone the native CMP-sialic acid synthetase gene insert (withoutthe decapeptide tag sequence) into plasmid CMPSIL-1, primer CMP5 (SEQ IDNO:1) in Table 4 was used for the forward primer and the primer shownbelow (SEQ ID NO:6) was used as the reverse primer in PCR as describedpreviously. Reverse Primer ##STR31##

The amplified PCR inserts and CMPSIL-1 plasmid were then digested withEco RI and Xba I (40 U/mg DNA) for one hour at 37° C. and the digestedDNA purified on 1 percent agrose gel. The purified native CMP-sialicacid synthetase gene insert and digested CMPSIL-1 plasmid were ligatedwith T4 DNA ligase (4U from Stratagene Co.) at 4° C. overnight (about 18hours) to form plasmid CMPSIL-W10.

The above procedure thus produced a second phagemid (CMPSIL-W10) fromCMPSIL-1 whose DNA was identical to that of CMPSIL-1 except for theabsence of a sequence encoding the decapeptide tag.

The plasmid CMPSIL-W10 was then transformed into E. coli Sure strain andplated on LB agar plates containing 250 mg/mL ampicillin. The positiveclones were selected by the assay of CMP-NeuAc synthesis activity aftergrowth on LB medium and induced with IPTG as discussed in Example 2. Theresulting E. coli Sure strain containing phagemid CMPSIL-W10 producednative or wild-type CMP-Sialic acid synthetase in an amount of about 35U/L.

The bacteria that harbored plasmids were grown on LB rich media (BactoTrypton, 25 g; yeast extract, 10 g; NaCl, 3 g; pH 7.0 in 1 L) containing250 mg/ml ampicillin to mid-logarithmic phase (OD₆₆₀ about 0.6-0.7) at37° C., and then induced with 0.5 mM IPTG (isopropylβ-D-thiogalactopyranoside) for 10 hours at 30° C. with shaking. Thecells were harvested by centrifugation (10,000×g 20 minutes, ) anddisrupted by a French , 4° C. pressure cell at 16,000 lb/in². The celldebris was removed by centrifugation at 23,000×g for 60 minutes and thesupernatant (cell free extract) was used for enzyme purification.

The cell free extract (30 mL) from 1 liter of culture was passed throughthe Orange A Dye column (1.5 mg/mL gel, 3 cm x 30 cm) and washed with200 mL of 0.2M Tris/HCl buffer containing 0.02M MgCl₂ and 0.2 mMdithiothreitol (DTT), pH 7.5. The enzyme was eluted with a lineargradient from zero to 1M KCl in the same buffer (total 200 mL). Theactive fractions were pooled and dialyzed in 2 L of 0.2M Tris/HCl buffer(pH 7.5) containing 0.02M MgCl₂ and 0.2 mM DTT. This enzyme preparationwas used for synthesis directly. The enzyme was further purified toabout 95 percent purity by FPLC using a Superose 12 HR 10/30 column fromPharmacia for use in kinetic studies.

2. Kinetic studies

The activity of the native or the modified CMP-NeuAc synthetase wasassayed using a thiobarbituric acid method described by Vann et al., J.Biol. Chem., 262:17556 (1987). Briefly, the enzyme was added to a 250 mLbuffer containing 5.5 mM CTP, 2.8 mM N-acetylneuraminic acid, 0.2M Tris,20 mM MgCl₂ and 0.2 mM DTT, pH 9.0, to form a mixture. After the mixturewas incubated at 37° C. for 30 minutes, 50 mL of 1.6M NaBH₄ was added todestroy excess NeuAc, and the mixture was heated at room temperature foran additional 15 minutes. The mixture was then put in the ice bath and50 mL of H₃ PO₄ were added to destroy NaBH₄.

The resulting mixture was kept at zero degrees C for five minutes thenincubated at 37° C. for 10 minutes to cleave the phosphoester bond ofthe formed CMP-NeuAc. The free NeuAc was oxidized with 50 mL of 0.2MNaIO₄ at room temperature for 10 minutes, and 400 mL of 4 percent NaAsO₂in 0.5 N HCl were added. The solution mixture was the transferred to atest tube containing 1 mL of 0.6 percent thiobarbituric acid in 0.5M Na₂SO₄, and heated in boiled water for 15 minutes. After the solution wascooled, 1 mL of the solution was taken out and mixed with 1 mL ofcyclohexanone. That mixture was shaken and centrifuged, and the upperlayer was taken for measurement at 549 nm (e=4.11 mM⁻¹ cm⁻¹).

Initial velocities were measured at various concentrations of CTP(1.25-5 mM) and NeuAc (2-8 mM) for kinetic studies. The data were fittedinto the sequential bi-bi substrate rate equation 3, shown below, toderive the Michaelis constants and maximum velocity (V) using a Sigmaplot program from Sigma Co. ##EQU1## where A is [CTP], B is [NeuAc],K_(a) and K_(b) are the Michael is constants for CTP and NeuAc,respectively, and K_(ia) is the dissociation constant (or inhibitionconstant) for CTP.

The specific activities and kinetic constants of the two enzymes werefound to be very similar. The native and the modified enzymes hadspecific activities of 2.1 U/mg and 2.3 U/mg, respectively. For thenative enzyme, the k_(cat) was 1.8 s⁻¹ and the K_(m) values for the twosubstrates, NeuAc and CTP, were 4 mM and 0.31 mM, respectively. Thetagged enzyme had a k_(cat) of 1.9s⁻¹ and K_(m) 's of 4.8 mM and 1.99mM, respectively, for NeuAc and CTP.

3. Enzyme stability

The native and modified enzymes were incubated at room temperature in a0.2M Tris buffer, pH 7.5, containing 0.02M MgCl₂ and 0.2 mMdithiothreitol. At defined time intervals, 30 mL aliquots were removedand assayed for activity as described above. Enzyme stabilities werestudied for a period of three days. The native enzyme has a half life ofabout 800 hours in a phosphate buffer (pH 7.5) at room temperature. Onthe other hand, the modified enzyme has a half life of about 80 hours,approximately 10 times less stable than the native, wild type.

4. pH profile

Both enzymes were assayed for the activity in 0.2M Tris buffers, from pH8 to pH 10.8, and in 50 mM sodium cacodylate buffers, from pH 4.5 to pH7.5. These buffers were prepared in the presence of 20 mM MgCl₂ and 6 mMMnCl₂ separately, containing 0.2 mM dithiothreitol. The assay solutioncontaining 5.5 mM CTP and 2.8 mM NeuAc was incubated at 37° C. for 30minutes, and the amounts of CMP-NeuAc formed were determined based onthe thiobarbituric acid assay.

The enzyme activities were studied at various pH values, from pH 4.5 to10.5, in the presence of two different metal ions, Mg²⁺ and Mn²⁺, whichwere known to affect the enzyme activity. Similar to the enzyme isolatedfrom the mammalian tissue, the native microbial CMP-sialic acidsynthetase was found to have an optimum pH at pH 7.5 in the presence ofMn²⁺, and at pH 9.5 in the presence of Mg²⁺. The tagged enzyme, however,showed an optimum activity at pH 9.5 in the presence of either Mg²⁺ orMn²⁺.

5. Substrate specificity

In 250 mL assay solution contained 2.8 mM of each substrate, 5.5 mM CTP,CMP-NeuAc synthetase and 0.2 M Tris buffer with 20 mM MgCl₂ and 0.2 mMDTT at pH 7.5 and pH 9.0. The incubation time varied from 15 minutes tofive hours depending on the activity of the enzymes toward the substrateanalogs. The formation of CMP-NeuAc derivatives was determined bythiobarbituric acid assay.

CMP-sialic acid synthetase from a variety of mammalian tissues was foundto be specific for CTP and sialic acids. It accepts some C-9 and C-8modified sialic acid analogs including fluorescent probes attached atthe 9-position. The enzyme from the mammalian system also accepts C-5modified substrates such as KDN and 5-N-glycolylneuraminic acid assubstrates. See, e.g. Shames et al., Glycobiology, 1:87 (1991); Auge etal., Tetrahed. Lett., 29:789 (1988); Kean et al., Methods Enzymol.,8:208 (1966); Roseman, S. Proc. Natl. Acad. Sci., 48:437 (1962); Grosset al., Eur. J. Biochem., 168:595 (1987); and Gross et at., Eur. J.Biochem., 117:583 (1988).

The results of substrate specificity studies for the native and modifiedrecombinant CMP-NeuAc synthetase enzymes are summarized in Table 5,below.

                                      TABLE 5                                     __________________________________________________________________________                             Tagged enzyme                                                                         Native enzyme                                                         pH 7.5                                                                            pH 9.0                                                                            pH 7.5                                                                            pH 9.0                                   __________________________________________________________________________        ##STR32##            1   1   1   1                                        201                                                                               ##STR33##            0.98                                                                              2.26                                                                              0.92                                                                              0.48                                     202                                                                               ##STR34##            0.92                                                                              1.7 0.95                                                                              0.46                                     203                                                                               ##STR35##            1.2 1.34                                                                              0.99                                                                              0.49                                     204                                                                               ##STR36##            1.1 1.2 0.98                                                                              0.52                                     205                                                                               ##STR37##            <0.05                                                                             <0.05                                                                             <0.05                                                                             <0.05                                    206                                                                               ##STR38##            <0.05                                                                             <0.05                                                                             <0.05                                                                             <0.05                                    207                                                                               ##STR39##            <0.05                                                                             <0.05                                                                             <0.05                                                                             <0.05                                    208                                                                               ##STR40##            <0.05                                                                             <0.05                                                                             <0.05                                                                             <0.05                                    209                                                                               ##STR41##            <0.05                                                                             <0.05                                                                             <0.05                                                                             <0.05                                    210                                                                               ##STR42##            <0.05                                                                             <0.05                                                                             <0.05                                                                             <0.05                                    __________________________________________________________________________

The cloned enzymes from E. coli system have similar substratespecificity to the enzyme from mammalian systems. Several sialic acidanalogs were synthesized and tested as substrates for the native and thetagged enzymes at pH 7.5 and pH 9.0.

Both enzymes have high activity for the C-9 modified sialic acid analogs(9-O-acetyl, 9-O-lactyl, 9-deoxy-9-fluoro, and 9-azido-9-deoxy NeuAc);however, the C-5 modified analogs (KDN, 5-deoxy KDN and5-deacetamido-5-epi-5-fluoro NeuAc) were not substrates. These resultssuggest that the 5-acetamido group of the sialic acids is critical forthe substrate recognition by the microbial enzymes.

Although both microbial enzymes have very similar substrate specificityat pH 7.5, they have different specificity at pH 9.0. At pH 7.5, thenative enzyme was found to be specific for NeuAc; the relative rates forthe C-9 modified NeuAc derivatives decreased to about 50 percent. Onthe-contrary, the relative rates of the C-9 modified analogs for thetagged enzyme are higher than that of N-acetylneuraminic acid (Table 5).

The C-5 and C-9 modified sialic acid derivatives used in this study wereprepared via sialic acid aldolase-catalyzed condensation with C-2 andC-6 modified N-acetylmannosamine (ManNAc) derivatives with pyruvate asset forth hereinafter in Example 10.

The 6-0-acylated ManNAc derivatives were prepared viatransesterification in DMF catalyzed by a subtilisin variant (8399 or8397) engineered to be stable in organic solvent. Zhong et al., J. Am.Chem. Soc., 113:683 (1991). The wild type enzyme (BPN') can also be usedbut requires more amount of enzyme as the variant is about 100 times(for 8399) to 1000 times (for 8397) more stable than the wild type. Itis thought that the enzymatic procedure described here for thepreparation of 6-O-acyl sugars is much more effective than the reportedchemical procedure. [For 9-O-acetyl-N-acetylneuraminic acid and9-O-lactyl N-acetylneuraminic acid: (a) Auge et al., Tetrahed. Lett.,25:4663 (1984) (6-O-acetyl-ManNAc was prepared chemically); (b) Kim etal., J. Am. Chem. Soc., 110:6481 (1988) (6-O-acetyl-ManNAc was preparedvia protease N reaction); (c) Auge et al., New J. Chem., 12:733 (1988)(for 9-O-lactyl-NeuAc from chemically synthesized 6-O-lactyl-ManNAc)];[For 9-azido-9-deoxy-N-acetylneuraminic acid: (d) Brossmer et al.,Biochem. Biophys. Res. Comm., 96:1282 (1980) (no physical data werereported)]; [For 3-deoxy-L-glycero-L-galactononulosonis acid (KDN): (e)Auge et al., Tetrahedron, 46:201 (1990)]; [For3,5-dideoxy-L-glycero-L-galactononulosonic acid (5-deoxy-KDN): (f) Augeet al., Tetrahed. Lett., 30:2217 (1989)].

Example 10

Preparation of sialic acid analogs

The C-5 and C-9 modified sialic acid derivatives used in the synthetasestudies described in Example 9 were prepared via sialic acidaldolase-catalyzed condensation with C-2 and C-6 modifiedN-acetylmannosamine (ManNAc) derivatives with pyruvate. The 6-O-acylatedManNAc derivatives were prepared via transesterification in DMFcatalyzed by a subtilisin variant (8399 or 8397) engineered to be stablein organic solvent. Zhong et al., J. Am. Chem. Soc., 113:683 (1991). Thewild type enzyme (BPN') can also be used but requires more amount ofenzyme as the variant is about 100 times (for 8399) to 1000 times (for8397) more stable than the wild type.

A. 9-O-Acetyl-N-acetylneuraminic acid (Compound 201)

In 2 mL of DMF were suspended 500 mg (2.2 mmol) of N-acetylmannosamine.Vinylacetate (1 mL, 5 equivalent) and 160 mg of subtilisin mutant 8399[Zhong et al., J. Am. Chem. Soc., 113:683 (1991)] were then added andthe suspension was stirred vigorously at room temperature. The reactionprogress was monitored by TLC with ethyl acetate/methanol (2/1). After 5hours when the formation of the di-acetylated derivatives began, thereaction was stopped by evaporating the vinylacetate and DMF. Methanolwas then added to dissolve the sugar. After the enzyme and salt werefiltered off, the filtrate was concentrated. The residual syrup waschromatographed on a silica gel, with ethyl acetate/methanol (10/1) togive 6-0-Acetyl N-acetylmannosamine (92 percent) as α/β mixture (3:1)based on the NMR spectrum.

α-Anomer:

¹ H NMR (D₂ O) δ1.98 (3H, s, NAc), 2.06 (3H, s, OAc), 3.58 (1H, dd, J=9,9 Hz, H-4), 3.96 (1H, dd, J=3.5, 9 Hz, H-3), 4.15-4.4 (4H, m,H-2,5,6,6), 5.04 (d, J=0.9 Hz, H-1a). α/β mixture ¹³ C NMR (D₂ O) δ23.1,24.7, 56.1, 56.7, 66.5, 66.6, 69.7, 69.8, 71.3, 72.4, 74.6, 76.6, 95.8,95.9, 176.7, 176.8, 177.6, 178.4. The product was identical to thatprepared with protease N reaction.

In a 10 mL of 0.1M potassium phosphate buffer (pH 7.5) were dissolved 10mM DTT, 0.5M pyruvate, 100 mg of 6-O-acetylmannosamine, prepared asdescribed above, and 1.5 mg NeuAc aldolase (36 U). The reaction mixturewas incubated at 37° C. for 8 days followed by lyophilization. Thelyophilized powder was dissolved in a small amount of water and directlyapplied to a Bio Gel P-2 column (3×90 cm) and eluted with a flow rate of6 mL/40 minutes at 4° C. To remove the trace amount of contaminatedNeuAc due to the hydrolysis of the product, another gel filtration wasrequired. Compound 201 was obtained in 47 percent yield. Its physicaldata were consistent with the reported data.

B. 9-0-Lactyl-N-acetylneuraminic acid (Compound 202)

ManNAc (200 mg) and 50 mg of subtilisin mutant 8399 were added to amixture of lactic acid ethyl ester 10 (4 mL) and 400 mL of 0.5 Nphosphate buffer (pH 7.5). The reaction mixture was shaken under 50° C.for three days. The solvent was then evaporated and methanol was addedto the residue. After the insoluble materials were filtered off, thefiltrate was concentrated. The residue was chromatographed on silica gelwith ethyl acetate/methanol (5/1) to give 6-O-Lactyl N-acetylmannosaminein 50 percent yield.

¹ H NMR (D₂ O) δ1.29 (3H, d, J=7 Hz,lactyl-CH₃), 1.91 (3H, s, OAc), 3.51(1H, dd, J=9.5, 9.5 Hz, H-4), 3.92 (1H, dd, J=9.5, 4.2 Hz, H-3), 4.16(1H, dd, J=1.8, 4.3 Hz, H-2), 4.2-4.4 (3H, m, H-5,6), 4.88 (d, J=0.9 Hz,H-1a), 4.97 (d, J=1.3 Hz, H-1b). ¹³ C NMR (D₂ O) δ20.10, 22.76, 22.93,54.17, 54.81, 65.1 67.6, 67.91, 69.43, 70.61, 93.87. HRMS calculated forC₁₁ H₁₉ NO₂ (M⁺): 293.1111. Found: 293.1091.

The procedure used to prepare Compound 202 was the same as that used toprepare Compound 201, except that 6-O-Lactyl N-acetylmannosamine wasused instead of 6-O-Acetyl N-acetylmannosamine. Another gel filtrationpurification was required to separate the product from NeuAc to giveCompound 202 in 18 percent yield. The physical data of the product wereconsistent with the reported values.

C. 7,9-Difluoro-7-epi-5-deaminoneuraminic acid (Compound 205)

Compound 205 was prepared using the aldolase reaction described hereinexcept that 4,6-dideoxy-4,6-difluoroglucose was used as the substrate.

HRMS: calcd 271. 0625; found, 271. 0649

¹ H-NMR (500 MHz, D₂ O) δ4.7 (m, H-7), 4.5 (dd, H-9, J_(H-) 9=47.5 Hz,J_(H-H) =3 Hz), 4.20 (td, H-8, J_(H-F) =18.5, J_(H-H) =3.0 Hz), 3.93 (m,H-6), 3.84 (m, H-4), 3.47 (t, H-5, J_(H-H) =10.5 Hz), 2.12 (dd, H-3e,J_(He-H4) =2.6 Hz, J_(He-H3) =12.5 Hz), 1.74 (dd, H-3a, J_(H-H4) =12.5,J_(H3a-H3e) =12 Hz). Peak assignment was accomplished by the use of 2Dtechnique.

D. 5-Epi-5-deamino-5-fluoro-neuraminic acid (Compound 206)

Compound 206 was prepared using the aldolase reaction described hereinexcept that 2-deoxy-2-fluoroglucose was used as the aldolase substrate.

HRMS: calcd. 269.0673; found, 269.0651. ¹³ C-NMR (D₂ O, CD₃ OD asstandard) δ174.5 (s, C-1), 96.4 (s, C-2), 34.5 (s, C-3), 65.5 (d, C-4,J_(C-4),F-5 =18 Hz, 91.2 (d, C5, J_(C-5),F-5 =191 Hz), 70.7 (d, C-6,J_(C-6),F-5 =18 Hz), 71.9 (s, C-7), 72.8 (s, C-8), 63.5 (s, C-9).

E. 3-Deoxy-L-glycero-L-galacto-2-nonulosonic acid (KDN) (Compound 207)

To a 10 mL potassium phosphate buffer (0.1M, pH 7.5) were added 10 mMDTT, 0.5M pyruvate, 0.1M D-mannose and 1.5 mg NeuAc aldolase. Thereaction mixture was shaken at 37° C. for three days. The reactionmixture was chromatographed with a Dowex-1 (HCO₃ ⁻) resin, eluted withzero to 1M ammonium bicarbonate gradient. The fractions containing KDNwere pooled and lyophilized three times repeatedly to remove thevolatile salt to give a 78 percent yield of KDN. The physical data ofthe product were are consistent with the reported.

F. 3,5-Dideoxy-L-glycero-L-galacto-2-nonulosonic acid (5-deoxy-KDN)(Compound 208)

2-Deoxyglucose (0.1M) was added to the solution instead of D-mannose,and the preparation procedure was the same as above. The reactionmixture was shaken for five days and Compound 208 was obtained in 30percent yield. The physical data of the product were consistent with thereported-data.

G. 9-(Dimethylphosphinyl)-9-deoxy-N-acetylneuraminic acid (Compound 209)

A solution of ManNAc (5.0 g, 2.6 mmol), Ac₂ O (10 mL) and pyridine (20mL) was stirred for 10 hours at room temperature, and the mixture wasconcentrated, followed by coevaporation with toluene. A solution of theresidue, benzyl alcohol (20 mL), BF₃.OEt₂ (1.6 g, 11.3 mmol) in CH₃ NO₂(150 mL) was gently refluxed for 2.5 hours. After cooling, the mixturewas concentrated. The residue was chromatographed on silica gel, withtoluene-EtOAc (1:2). The isolated benzyl2-acetamido-4,5,6-tri-O-acetyl-2-deoxy-a-D-mannopyranoside and 0.15 g ofNaOMe was dissolved in MeOH (100 mL) and the solution was stirred for 30minutes at room temperature, and neutralized by addition of Dowex 50W-X8(H⁺). After the resin was filtered off, the filtrate was concentrated,followed by coevaporation with pyridine. A solution ofdimethylphosphinic chloride (1 g, 8.8 mmol) in DMF was added to a coldsolution of benzyl 2-acetamido-2-deoxy-a-D-mannopyranoside (0.50 g, 1.6mmol), 2,6-lutidine (0.34 g, 3.2 mmol) in anhydrous DMF (30 mL) in a dryice-acetone bath, and the reaction was allowed to slowly warm to roomtemperature. The reaction was monitered by TLC with (1M NH₄OAc/2-proponal/EtOAc, 1/2.4/3.4). After 10 hours, the reaction mixturewas directly applied to silica gel chromatography, eluted with CHCl₃/EtOAc/MeOH (5/2/1) to give benzyl2-acetamido-2-deoxy-6-(dimethyl-phosphinyl)-a-D-mannopyranoside (56percent yield).

¹ H NMR (D₂ O) δ1.46 (3H, d, J=13.4 Hz, P-CH₃), 1.5 (3H, d, J=13.4 Hz,P-CH₃), 1.89 (3H, s, NAc), 3.53 (1H, dd, J=8, 8 Hz, H-4), 3.76 (1H, dd,J=8, 4.3 Hz, H-3), 4.0 (3H, m, H-5, 6), 4.18 (1H, d, J=4.3 Hz, H-2),4.44 (1H, d, J=9.2 Hz, Bn-H-1a), 4.55 (1H, d, J=9.2 Hz, Bn-H-1b), 4.76(1H, s, H-1), 7.28 (5H, s, Bn).

A solution of benzyl2-acetamido-2-deoxy-6-(dimethyl-phosphinyl)-α-D-mannopyranoside,prepared as described above, (100 mg, 0.26 mmol) in ethanol/water (10mL; 1/1) was hydrogenated with 50 mg 10 percent Pd/C for 10 hours. Thereaction progress was monitered with TLC (EtOAc/AcOH/H₂ O, 8/2/1). Thecatalyst was filtered and the filtrate was concentrated to give2-acetamido-2-deoxy-6-(dimethylphosphinyl)-α-D-mannopyroside (100percent yield).

¹ H NMR (D₂ O) δ1.51 (6H, d, J=13.6 Hz) 1.94 (3H, s, NAc), 3.45 (2H, m),3.9 (1H, dd, J=8,4.3 Hz, H-3), 4.1 (3H, m, H-5,6), 4.87, 4.98 (1H, s,H-1).

Sialic acid aldolase catalyzed aldol condensation of2-acetamido-2-deoxy-6-(dimethylphosphinyl)-α-D-mannopyroside, preparedas described above, and pyruvic acid was conducted for four days. Theproduct was purified with a Bio Gel P-2 at 4° C. to give Compound 209 in42 percent yield.

¹ H NMR (D₂ O) δ1.48 (6H, d, J=14.2 Hz, P-CH₃), 1.67 (1H, dd, J=11,13Hz, H-3ax), 1.9 (3H, s, NAc), 2.08 (1H, dd, J=5.3, 13 Hz, H-2eq), 3.45(1H, d, J=9.3Hz, H-7), 3.8-4.0 (6H, m, H-4,5,6,8,9).

H. 9-Azido-9-deoxy-N-acetylneuraminic acid (Compound 203)

A solution of ManNAc (5.0 g, 2.6 mmol), and Ac₂ O (10 mL) in pyridine(20 mL) was stirred for 10 hours at room temperature, and the mixturewas concentrated, followed by coevaporation with toluene. A solution ofthe residue, allyl alcohol (2.63 g, 45.2 mmol; 3.1 mL), BF₃ •OEt₂ (1.60g, 11.3 mmol; 1.39 mL) in CH₃ NO₂ (150 mL) was gently refluxed for 2.5hours. After cooling, the mixture was concentrated. The residue waschromatographed on silica gel, with toluene-EtOAc (1:2) to give allyl2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-mannopyranoside in 6.47 g (74percent);

¹ H NMR (CDCl₃) δ1.99, 2.05, 2.06, 2.12 (3H, s, 3 x OAc, NHAc),4.00-4.03 (1H, m, H-5), 4.07 (1H, dd, J=2.45, 12.24 Hz, H-6a), 4.29 (1H,dd, J=5.34, 12.23 Hz, H-6b), 4.63 (1H, ddd, J=1.43, 4.60, 9.11 Hz, H-2),4.81 (1H, d, J=1.43 Hz, H-1), 5.11 (1H, t, J=10.18 Hz, H-4), 5.36 (1H,dd, J=4.59, 10.19 Hz, H-3), 5.82 (1H, d, J=9.11 Hz, NHAc); ¹³ C NMR(CDCl₃) δ20.67, 20.74, 23.33, 50.29 , 62.42, 66.05, 68.02, 68.64, 69.11,98.02, 118.42, 132.83, 169.89, 169.96, 170.08, 180.55.

A solution of allyl2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-mannopyranoside, prepared asdescribed above, (6.46 g, 16.7 mmol) and methanolic NaOMe (2 mL; 1Msolution) in MeOH (100 mL) was stirred for two hours at roomtemperature, and neutralized by addition of Dowex 50W-X8 [H⁺ ]. Afterthe resin was filtered off, the filtrate was concentrated followed bycoevaporation with pyridine. A solution of tolylsulfonyl chloride (3.50g, 18.3 mmol) in pyridine (20 mL) and CH₂ Cl₂ (30 mL) was added dropwiseto a cooled solution of the residue in pyridine (30 mL) and CH₂ Cl₂ (50mL) at 0°-5° C. over 30 minutes, and the mixture was stirred for 10hours at room temperature then cooled. Acetic anhydride (30 mL) wasadded to the mixture, and the mixture was stirred for five hours roomtemperature. The mixture was concentrated, and the residue waschromatographed on silica gel, with toluene EtOAc (1:2), to give allyl2-acetamido-3,4-di-O-acetyl-2-deoxy-6-O-tolylsulfonyl-α-D-mannopyranoside(3.68 g, 44%);

¹ H NMR (CDCl₃) δ1.98 (6H, s, 2 x Ac), 2.04 (3H, s, Ac), 2.46 (3H, s,CH₃ of tosyl), 3.96-4.00 (1H, m, H-4), 4.10 (1H, dd, J=4.50, 11.50 Hz,H-6a), 4.30 (1H, dd, J=2.00, 11.50 Hz, H-6b), 4.62 (1H, ddd, J=1.50,4.50, 9.00 Hz, H-2), 4.78 (1H, d, J=1.50, 4.50, 9.00 Hz, H-2), 4.78 (1H,d, J=1.50 Hz, H-1), 5.16 (1H, t, J=10.0 Hz, H-4), 5.33 (1H, dd, J=4.50,10.0 Hz, H-3), 6.03 (1H, d, J=9.50 Hz, NHAc); ¹³ C NMR (CDCl₃) δ20.59,20.78, 21.64, 23.26, 50.00, 65.75, 67.99, 68.10, 68.69, 69.20, 98.04,118.37, 127.89, 129.89, 132.79, 170.28. HRMS calcd. for C₂₂ H₂₉ NO₁₀ S(M⁺): 500.1590. Found: 500.1590.

A solution of allyl2-acetamido-3,4-di-O-acetyl-2-deoxy-6-O-tolylsulfonyl-α-D-mannopyranoside,prepared as described above, (3.68 g, 7.37 mmol) and NaI (2.21 g, 14.7mmol) was gently refluxed for 10 hours, and cooled. The mixture wasconcentrated, and the residue was chromatographed on silica gel, withtoluene-EtOAc (1:2), to give allyl2-acetamido-3,4-di-O-acetyl-2,6-dideoxy-6-iodo-α-D-mannopyranoside (2.96g, 88 percent);

¹ H NMR (CDCl₃) δ1.99, 2.05, 2.08 (3H, s, 2 x OAc, NHAc), 3.19 (1H, dd,J=7.50, 11.0 Hz, H-6a), 3.35 (1H, dd, J=3.0, 11.0 Hz, H-6b), 3.71 (1H,ddd, J=3.0, 7.5, 10.0 Hz, H-5), 4.63 (1H, ddd, J=1.5, 4.5, 9.5 Hz, H-2),4.82 (1H, d, J=1.5 Hz, H-1), 4.98 (1H, t, J=10.0 Hz, H-4), 5.36 (1H, d,J=4.5, 10.0 Hz, H-3), 5.79 (1H, d, J=9.5, NHAc); ¹³ C NMR (CDCl₃) δ5.49,20.74, 23.34, 50.17, 68.51, 69.11, 70.07, 97.76, 118.54, 132.75, 169.93,169.99. HRMS calcd for C₁₅ H₂₂ NO₇ I (M⁺): 456.0519. Found: 456.0520.

0.9 Grams of allyl2-acetamido-3,4-di-O-acetyl-2,6-dideoxy-6-iodo-α-D-mannopyranoside,prepared as described above, was dissolved in 10 mL of DMF, 3equivalents of NaN₃ were added and the reaction mixture was heated at100° C. for 10 hours.

The product exhibited the same Rf value (0.7) as the iodo derivative onTLC (ethyl acetate). However on the TLC, the azido derivative was UVinvisible, whereas the iodo derivative was UV visible.

After evaporating of the solvent, the residue was directly applied tothe silica gel column chromatography, (hexane/ethyl acetate, 3/2) togive allyl2-acetamido-3,4-di-O-acetyl-6-azido-2,6-dideoxy-α-D-mannopyranoside (0.5g, 69 percent);

¹ H NMR (CDCl₃) δ1.92 (3H, s, NHAc), 1.98 (3H, s, OAc), 1.99 (3H, s,OAc), 3.2 (1H, dd, J=3, 13 Hz, H-6a), 3.27 (1H, dd, J=6.5, 13 Hz, H-6b),3.87 (1H, ddd, J=3, 6.5, 11.7 Hz, H-5), 3.95 (1H, dd, J=6, 18 Hz,allyl-H-1a), 4.11 (1H, dd, J=6. 18 Hz, allyl-H-1b), 4.54 (1H, ddd, J=2,5.5, 9.8 Hz, H-2), 4.74 (1H, d, J=2 Hz, H-1), 5.02 (1H, dd, J=9.8, 9.8Hz, H-4), 5.14 (1H, dd, J=2, 11.3 Hz, allyl-H-3a), 5.23 (1H, dddd, J=6,6, 11.3, 16 Hz, allyl-H-2), 6.17 (1H, d, J=9.8 Hz, NH); ¹³ C NMR (CDCl₃)δ20.8, 23.50, 67.68, 68.4, 69.2, 97.5, 117.8, 132.4, 169.8, 170.1.

Allyl2-acetamido-3,4-di-O-acetyl-6-azido-2,6-dideoxy-α-D-mannopyranoside,prepared as described above, (0.5 rag) was dissolved in 10 mL methanolcontaining 0.2M MeONa. After five minutes, Dowex 50 cation exchangeresin was added to the mixture to neutralize. The resin was filtered andthe filtrate was concentrated to yield allyl2-acetamido-6-azido-2,6-dideoxy-α-D-mannopyranoside (99 percent).

¹ H NMR-(CDCl₃) δ2.03 (3H, s, NAc), 3.4-3.5 (2H, m, H-6a, 6b), 3.59 (1H,dd, J=9.8, 9.8 Hz, H-4), 3.75 (1H., m, H-5), 4.02 (1H, dd, J=9.8, 5 Hz,H-3), 4.18, 4.39 (1H, dd, J=5, 8.1 Hz, H-2), 4.74 (1H, br-s, OH), 4.8(1H, s, H-1), 4.87 (1H, br-s, OH), 5.23 (1H, dd, J=1.2, 10 Hz,allyl-H-3a), 5.32 (1H, dd, J=17, 1.2 Hz, allyl-H-3b), 5.90 (1H, dddd,J=6, 6, 10, 17 Hz, allyl-H-2), 6.69 (1H, d, J=8.1, NH).

A suspension of allyl2-acetamido-6-azido-2,6-dideoxy-α-D-mannopyranoside (200 mg), 1.2equivalents PdCl₂ and 2.4 equivalents of NaOAc were dissolved in 95percent acetic acid (5 mL). The reaction was stirred at room temperatureovernight and concentrated. The residue was purified with silica gelchromatography, (CHCl₃ /ethyl acetate/methanol, 5/2/2), to give2-acetamido-6-azido-2,6-dideoxy-α-D-mannopyranoside in 31 percent yield.

¹ H NMR (D₂ O) δ1.66 (1H, 3H, s, NAc), 3.46 (1H, dd, J=9.8, 9.8 Hz,H-4), 3.45-3.55 (2H, m, H-6a, 6b), 3.82 (1H, m, H-5), 3.87 (1H, dd,J=4.6, 9.8 Hz, H-3), 4.15 (1H, d, J=4.6 Hz, H-2), 4.88 (d, J=1.2 Hz,H-1b), 4.97 (s, H-1a).

In a 10 mL of 0.1M potassium phosphate buffer (pH 7.5) containing 10 mMDTT and 0.5M pyruvate were dissolved 50 mg of6-azido-6-deoxy-N-acetylmannosamine and 1.5 mg of NeuAc aldolase. Thestarting material was consumed in 14 hours. The solution was lyophilizedand the purification was carried out with Bio Gel P-2 gel filtration(3×90 cm) chromatography with a flow rate of 6 mL/40 minutes, at 4° C.The fractions containing the product were pooled and freezed dry to giveCompound 203 in 84 percent yield.

¹ H NMR (D₂ O) δ1.66 (1H, dd, J=1, 13 Hz, H-3ax), 1.89 (3H, s, NAc),2.05 (1H, dd, J=4.4, 13 Hz, H-eq), 3.31 (1H, dd, J=5.8, 12 Hz, H-9a),3.37 (1H, dd, J=1.2, 10 Hz, H-7), 3.45 (1H, dd, J=3.3, 12 Hz, H-9b),3.7-3.9 (4H, m, H-4, 5, 6, 8). HRMS calcd for C₁₁ H₁₈ N₄ D₈ (M-H⁻):333.1046. Found: 333.1046.

I. 9-Deoxy-9-fluoro-N-acetylneuraminic acid (Compound 204)

A solution of allyl 2-acetamido-2-deoxy-α-D-mannopyranoside, prepared asdescribed above, 2.0 g, and 1.2 equivalents of tritylchloride wasstirred for 10 hours at 72° C. After the reaction mixture was cooled tozero degrees C, 2.5 equivalents of benzoyl chloride were added to themixture. The reaction mixture was allowed to slowly warm to roomtemperature in two hours. After the reaction was completed, ice waterwas added, and the reaction mixture was extracted with ethyl acetate.The organic extracts were washed with 1N HCl twice, dried andconcentrated. The residue was applied to silica gel chromatography,eluted with hexane/ethyl acetate (10/1) to give allyl2-acetamido-3,4-di-O-benzoyl-2-deoxy-6-O-trityl-α-D-mannopyroside (23percent yield).

¹ H NMR (CDCl₃) δ2.05 (3H, s, NAc), 3.87 (1H, dd, J=9.8, 9.8 Hz, H-4),4.05 (1H, dd, J=6, 12 Hz, allyl), 4.1 (3H, m, H-5,6,6'), 4.2 (1H, m,allyl), 4.96 (1H, d, J=11.3 Hz, allyl), 5.07 (1H, d, J=11.3 Hz, Allyl),5.17 (1H, m, allyl), 5.97 (1H, d, J=7.4 Hz, N HAc).

A suspension of 1.5 g of allyl2-acetamido-3,4-di-O-benzoyl-2-deoxy-6-O-trityl-α-D-mannopyroside,prepared as described above, in 80 percent acetic acid (10 mL) wasallowed to stirred for overnight at room temperature. After the reactionmixture was concentrated, the residue was applied to silica gelchromatography, eluted with hexane/ethyl acetate (5/1) to give allyl2-acetamido-3,4-di-benzoyl-2-deoxy-α-D-mannopyranoside (90 percentyield).

¹ H NMR (D₂ O) δ2.0 (3H, s, NHAc), 3.78 (2H, m, H-5,6), 4.04 (1H, m,H-6), 4.1 (1H, m, allyl), 4.23 (1H, ddd, J=1.26, 5.73, 12.7 Hz, allyl),4.88 (1H, dd, J=4.56, 9.24 Hz, allyl), 4.93 (1H, d, J=1.06 Hz, H-1),5.24 (1H, dd, J=1.25, 10.5 Hz, allyl), 5.33 (1H, dd, J=1.4, 17.2 Hz,allyl), 5.63 (1H, dd, J=10.1, 10.1 Hz, H-4), 5.91 (1H, dd, J=4.55, 10.34Hz, H-3), 5.95 (1H, m, allyl), 6.83 (1H, d, J=9.15 Hz, HNAc).

To a stirred solution of (diethylamino)sulfur trifluoride (0.5 mL) indry diglyme (2mL) was added a solution of allyl2-acetamido-3,4-di-benzoyl-2-deoxy-α-D-mannopyranoside, prepared asdescribed above, (100 mg) in dry diglyme (3 mL) at room temperature, andthe reaction mixture was stirred for one hour at room temperature andthree hours at 40° C. After the starting material was consumed, thereaction mixture was poured onto ice-water and extracted with ethylacetate. The extract was dried, concentrated, and the residue wasapplied to silica gel chromatography. After the impurity was eluted withhexane, the product was eluted with ether to give the fluorinatedproduct in 89 percent yield. The product was then dissolved in 5 mL of1N sodium methoxide in methanol to remove benzoyl group. After 20minutes, Dowex 50W X-8[H⁺ ] was added to neutralize the reactionmixture. The resin was filtered and the filtrate was concentrated togive allyl 2-acetamido-2,6-dideoxy-6-fluoro-α-D-mannopyranoside in 99percent yield. The product (50 mg), 1.2 equivalents of palladium (II)acetate, and 2.5 eq of sodium acetate in 95 percent acetic acid (5 mL)were stirred at 50° C. for 18 hours, and the solvent was removed undervacuum. The residue was applied to silica gel chromatography, elutedwith ethyl acetate/methanol (2/1) to obtain2-acetamido-2,6-di-deoxy-6-fluoro-α-D-mannopyranoside in 73 percentyield.

¹ H NMR (D₂ O) δ1.9 (3H, s, NAc), 3.5 (1H, dd, J=10.3, 10.3 Hz, H-4),3.72 (1H, m, H-5), 3.93 (1H, dd, J=4.5, 10.3 Hz, H-3), 4.16 (1H, d,J=4.5 Hz, H-2), 4.46 (2H, m, H-6), 4.9, 5.0 (1H, s, H-1).

A solution of 2-acetamido-2,6-di-deoxy-6-fluoro-α-D-mannopyranoside,prepared as described above, (20 mg) and pyruvic acid sodium salt (255mg, 30 equivalents) in 0.1M potassium phosphate buffer (pH 7.5, 10 mL)in the presence of N-acetylneuraminic acid aldolase (100 U) wasincubated at 37° C. for 8 days. The reaction mixture was lyophilized andchromatographed with Bio Gel P-2 column to give Compound 204 in 22percent yield. The physical data were in accordance with reportedvalues. Sharma et al., Carb. Res., 175:25 (1988).

The foregoing is intended as illustrative of the present invention butnot limiting. Numerous variations and modifications may be effectedwithout departing from the true spirit and scope of the novel conceptsof the invention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 10                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATATTGAATTCTAAACTAGTCGCCAAGGAGACAGTCATAATGAGAACAAAAATTATTGCG60                (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 67 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GCGCTCTAGACTATTAAGAACCGTAGTCCGGAACGTCGTACGGGTATTTAACAATCTCCG60                CTATTTC67                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TyrProTyrAspValProAspTyrAlaSer                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ATATTGAATTCAGAAGGAGATATACATATGAGAACAAAAATTATT45                               (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GCGCAAGCTTCATTTAACAATCTCCG26                                                  (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GCGCTCTAGACTATTATTTAACAATCTCCGCTATT35                                         (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GCGCAGGCCGCTGAATTGACCGGAGGGGCCCGGCCGCCG39                                     (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AlaGlnAlaAlaGluLeuThrGlyGlyAlaArgProPro                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GAATTCTAAACTAGTCGCCAAGGAGACAGTCATAATG37                                       (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TyrProTyrAspValProAspTyrAlaSer                                                1510                                                                          __________________________________________________________________________

We claim:
 1. E. coli transformed with phagemid CMPSIL-1.
 2. PhagemidCMPSIL-1.
 3. E. coli transformed with phagemid CMPSIL-W10.
 4. PhagemidCMPSIL-W10.